A chemoenzymatic approach toward the identification of fucosylated glycoproteins and mapping of N-glycan sites.

Fucose (Fuc)-containing glycoconjugates play important roles in numerous physiological and pathological processes. Given the biological importance of post-translational glycosylation, a specific and robust strategy for the identification of fucosylated glycoproteins is highly desirable. In this study, we demonstrate an alternative way of labeling of fucosylated structures by metabolic engineering, using a chemoenzymatic approach. In this approach, the activities of Bacteroides fragilis 9343 L-fucokinase/guanosine-5'-diphosphate-Fuc pyrophosphorylase and human α1,3-fucosyltransferase 9 are combined in a Namalwa cellular model. Interestingly, this system could be applied to labeling of alkyne-modified fucosylated glycoproteins. N-Glycan site mapping and identification were done using an in vitro selective chemical ligation reaction and isotope-coded glycosylation site-specific tagging, subsequent to liquid chromatography-tandem mass spectrometry analysis. This work illustrates the use of a click chemistry-based strategy combined with a glycoproteomic technique to get further insight into the pattern of Fuc-mediated biological processes and functions.