Glycosyltransferase enzymatic assays: Overview and comparative analysis

Glycosyltransferases (GTs) are enzymes that catalyze the transfer of an activated sugar donor to a variety of acceptors including proteins, lipids, carbohydrates, and other small molecules. GTs participate in numerous cellular and physiological processes in both prokaryotic and eukaryotic cells. Those include prokaryotic cell wall biogenesis, eukaryotic post-translational protein modifications, extracellular matrix synthesis, cell signaling, biofilm formation and many others. As such, GTs are exploited as molecular therapeutical targets but also as synthetic tools for the development of polysaccharides and glycoconjugates. In vitro study of GTs activities is now essential to characterize the growing number of predicted GTs, available from sequenced genomes, in order to determine their specificities, their modes of action and their roles in vivo. However, characterization of glycosyltransferases in vitro, both on cellular extracts and on purified enzymes, faces significant challenges. Many methods are currently employed i. e. radiochemical techniques, spectrometric measurements, generally after coupling with∗ other reactions, and even more sophisticated strategies involving product separations by chromatography or/and electrophoresis, followed by detailed structural analysis by NMR or mass spectrometry. Here we overview the common methods deployed for the characterization of GTs. We highlight the challenges arising from these enzymes. The advantages and limitations of each of the presented techniques are also discussed.