Chemical labeling and analysis of sialoglycans

Abstract

Every cell is covered with a dense layer of glycans, which is termed glycocalyx. The outermost monosaccharide of these glycans is often sialic acid, which are a family of nine-carbon monosaccharides. Sialylated glycans play important roles in various physiological and pathological processes, including pathogen invasion, cancer progress, and immune response. Lectins and antibodies have long been used for labeling and detecting sialylated glycans. However, they suffer from low affinity and poor specificity. In the past decades, metabolic glycan labeling (MGL) has emerged as a powerful tool for probing sialylation in live cells and living animals. In the MGL strategy, unnatural sugars containing a bioorthogonal functional group is metabolically incorporated into sialylated glycans by exploiting the underlying glycan biosynthetic pathway. Subsequent bioorthogonal reactions enable conjugation of sialylated glycans with imaging probes or enrichment tags. In this review, we first give a brief introduction on the history and current progress of MGL and bioorthogonal chemistry. Secondly, we discuss the application of MGL in the studies of dynamic changes and regulation of sialylation in various biological processes. Several examples from our research group on the studies of sialylation dynamics in epithelial-mesenchymal transition (EMT), neuronal stem cell differentiation, and cardiac hypertrophy are presented. By metabolic labeling of sialoglycans with ManNAz during EMT, we observed that cellular sialylation was downregulated during EMT, followed by reversion and upregulation. In neuronal stem cells, metabolic labeling of cell-surface sialoglycans has been exploited to discover cell-surface markers. Sialylation in cardiac hypertrophy was investigated by MGL in intact rat hearts, which revealed upregulation of sialylation during hypertrophy. Thirdly, aiming to expand the applications of MGL, we have developed new methods for protein-specific glycan imaging, cell- and tissue-selective glycan labeling, glycan Raman imaging, and novel unnatural sugars. By combining MGL with site-specific protein labeling, we developed a cis-membrane FRET-based methodology to visualize glycans of specific proteins of interest on live cells. Using the protein-specific glycan imaging technique, we found that sialylation of αXβ2 integrin is important for its activation. To overcome the limitation that MGL lacks cell-type selectivity, we developed the liposome-assisted bioorthogonal reporter (LABOR) strategy, in which azido sialic acid is encapsulated in ligand-targeted liposomes for cell-selective labeling of sialylated glycans via the receptor-mediated endocytosis. We demonstrated the LABOR strategy by selective labeling of sialoglycans in folate receptor-overexpressed cells. LABOR enables targeted imaging of tumor-associated sialoglycans and brain sialoglycans in living mice. Fourthly, novel unnatural sialic acids have been developed for probing specific sialoglycoforms, identifying sialic acid-binding proteins, remodeling cell-surface sialic acid, and bioorthogonal Raman imaging of sialylated glycans. Finally, we give an outlook on future directions of studying sialobiology using these chemical methods.