Cell-type-specific labeling and profiling of glycans in living mice

Metabolic labeling of glycans with clickable unnatural sugars has enabled glycan imaging and glycoproteomics in mixed cell populations. However, in vivo labeling and profiling of cell-type-specific glycans remains challenging. Here, we develop genetically encoded metabolic glycan labeling (GeMGL), a cell-type-specific strategy based on a bump-hole pair of an unnatural sugar and its matching engineered enzyme. N pentynylacetylglucosamine (GlcNAl) serves as a bumped analog of N acetylglucosamine (GlcNAc) that can be specifically incorporated into glycans of cells expressing an engineered mutant of UDP-GlcNAc pyrophosphorylase, AGX2F383G. GeMGL with the 1,3-di-O-propionylated GlcNAl (1,3-Pr2GlcNAl) and AGX2F383G pair is demonstrated in cell co-cultures, and used for specific labeling of glycans in mouse xenograft tumors. By generating a transgenic mouse line with AGX2F383G expressed under cardiomyocyte-specific promoter, we perform specific imaging of cardiomyocyte glycans in the heart and identified 582 cardiomyocyte O-GlcNAcylated proteins with no interference from other cardiac cell types. GeMGL will facilitate cell-type-specific glycoproteomics in various tissues and disease models.