Applying transcriptomics to studyglycosylation at the cell type level.

The complex multi-step process of glycosylation occurs in a single cell, yet current analytics generally cannot measure the output (the glycome) of a single cell. Here, we addressed this discordance by investigating how single cell RNA-seq data can be used to characterize the state of the glycosylation machinery and metabolic network in a single cell. The metabolic network involves 214 glycosylation and modification enzymes outlined in our previously built atlas of cellular glycosylation pathways. We studied differential mRNA regulation of enzymes at the organ and single cell level, finding that most of the general protein and lipid oligosaccharide scaffolds are produced by enzymes exhibiting limited transcriptional regulation among cells. We predict key enzymes within different glycosylation pathways to be highly transcriptionally regulated as regulatable hotspots of the cellular glycome. We designed the Glycopacity software that enables investigators to extract and interpret glycosylation information from transcriptome data and define hotspots of regulation.

Display of the human mucinome with defined O-glycans by gene engineered cells.

Mucins are a large family of heavily O-glycosylated proteins that cover all mucosal surfaces and constitute the major macromolecules in most body fluids. Mucins are primarily defined by their variable tandem repeat (TR) domains that are densely decorated with different O-glycan structures in distinct patterns, and these arguably convey much of the informational content of mucins. Here, we develop a cell-based platform for the display and production of human TR O-glycodomains (~200 amino acids) with tunable structures and patterns of O-glycans using membrane-bound and secreted reporters expressed in glycoengineered HEK293 cells. Availability of defined mucin TR O-glycodomains advances experimental studies into the versatile role of mucins at the interface with pathogenic microorganisms and the microbiome, and sparks new strategies for molecular dissection of specific roles of adhesins, glycoside hydrolases, glycopeptidases, viruses and other interactions with mucin TRs as highlighted by examples.