Use of metabolic glycoengineering and pharmacological inhibitors to assess lipid and protein sialylation on cells

Abstract

Metabolic glycoengineering (MGE) has been developed to visualize carbohydrates on live cells. The method allows the fluorescent labeling of sialic acid (Sia) sugar residues on neuronal plasma membranes. For instance, the efficiency of glycosylation along neurite membranes has been characterized as cell health measure in neurotoxicology. Using human dopaminergic neurons as model system, we asked here, whether it was possible to separately label diverse classes of biomolecules and to visualize them selectively on cells. Several approaches suggest that a large proportion of Sia rather incorporated in non-protein components of cell membranes than into glycoproteins. We made use here of deoxymannojirimycin (dMM), a non-toxic inhibitor of protein glycosylation, and of N-butyl-deoxynojirimycin (NBdNM) a well-tolerated inhibitor of lipid glycosylation, to develop a method of differential labeling of sialylated membrane lipids (lipid-Sia) or sialylated N-glycosylated proteins (protein-Sia) on live neurons. The time resolution at which Sia modification of lipids/proteins was observable was in the range of few hours. The approach was then extended to several other cell types. Using this technique of 'target-specific MGE', we found that in dopaminergic or sensory neurons > 60% of Sia is lipid bound, and thus polysialic acid-neural cell adhesion molecule (PSA-NCAM) cannot be considered the major sialylated membrane component. Different from neurons, most Sia was bound to protein in HepG2 hepatoma cells or in neural crest cells. Thus, our method allows visualization of cell-specific sialylation processes for separate classes of membrane constituents.