Localized Chemical Remodeling for Live Cell Imaging of Protein‐Specific Glycoform

Abstract

Live cell imaging of protein‐specific glycoforms is important for the elucidation of glycosylation mechanisms and identification of disease states. The currently used metabolic oligosaccharide engineering (MOE) technology permits routinely global chemical remodeling (GCM) for carbohydrate site of interest, but can exert unnecessary whole‐cell scale perturbation and generate unpredictable metabolic efficiency issue. A localized chemical remodeling (LCM) strategy for efficient and reliable access to protein‐specific glycoform information is reported. The proof‐of‐concept protocol developed for MUC1‐specific terminal galactose/N‐acetylgalactosamine (Gal/GalNAc) combines affinity binding, off‐on switchable catalytic activity, and proximity catalysis to create a reactive handle for bioorthogonal labeling and imaging. Noteworthy assay features associated with LCM as compared with MOE include minimum target cell perturbation, short reaction timeframe, effectiveness as a molecular ruler, and quantitative analysis capability.

Glycoenzyme Tool Development: Principles, Screening Methods, and Recent Advances†

“…To ensure the spatial specificity, the enzyme is caged during the enzyme anchoring step, and then activated in situ to perform confined enzymatic oxidation. [ 129‐132 ] Utilization of these above‐mentioned methods will open up new possibilities in high‐throughput screening in glycoenzyme directed evolution.…”

Recent Advances in the Chemical Biology of N-Glycans

“…In addition to glycan function analysis, the therapeutic application of metabolic glycan labeling is being vigorously investigated [ 176 ]. Glycan engineering by chemical [ 177 , 178 ] and chemoenzymatic [ 179 , 180 , 181 , 182 , 183 ] methods has also been investigated. In addition, de novo glycans on cell surfaces have also been reported, such as the direct introduction of defined glycan structures into plasma membranes by lipid insertion, liposomal fusion, and tag technology [ 184 , 185 , 186 , 187 , 188 , 189 ].…”

Site-Selective Palladium-Catalyzed Oxidation of Glucose in Glycopeptides

“…The resulting carbonyl group can then be readily transformed into a hydrazone or oxime. 15 Enzymatic oxidation [16][17][18][19][20] has been reported for galactose, mannose, GalNAc, and GlcNAc, and has been applied to the diversification of glycopeptide antibiotics and the introduction of amines and amides. 21,22 In addition, oxidation and subsequent oxime/hydrazone formation can also be used as a strategy to introduce biotin or fluorophores (also on-cell).…”