Visualizing metabolically-labeled glycoconjugates of living cells by strain-promoted alkyne-azide cycloaddition by cu-free click reactions

The bioorthogonal chemical reporter strategy is emerging as a versatile methodology for labeling biomolecules such as nucleic acids, lipids, carbohydrates and proteins. In particular, a biomolecule modified by an azido group can be tagged by Staudinger ligation using modified phosphines, by a copper (I)-catalyzed cycloaddition with terminal alkynes (CuAAC), or by a strainpromoted alkyne-azide cycloaddition (SPAAC). The latter ligation has as a major advantage that it does not require a cytotoxic metal catalyst. We have developed 4 dibenzocyclooctynol (DIBO) and derivatives thereof as convenient reagents for quantification and visualization of metabolically labeled cell-surface glycoconjugates of living cells by SPAAC. The methodology has been validated by comparing the extend of metabolic labeling in a group of established CHO glycosylation mutant cells (Lec2, Lec 13, Lec 32, Cog-1, and Cog-2) with traditional lectin staining using FITC-peanut agglutinin (PNA) and FITC-Ricinus communis-I (RCA-I) with or without Vibrio cholerae sialidase treatment. In addition to cell surface glycoproteins, we have shown that the technology can be employed to visualize the fibrillar network deposited by adherent fibroblast cultures. Furthermore, we demonstrated clear differences in the trafficking and turnover of sialylated molecules in wild type human fibroblasts and fibroblasts from patients and animal models with lysosomal storage disorders such as mucolipidosis II and lysosomal neuramindase (Neu1) deficiency. Together, these results suggest that this methodology will represent a valuable tool in the analysis of cells with altered biosynthesis, trafficking and turnover of glycoproteins and extracellular matrix proteins.