The overall glycome of the cell is made up of a variety of glycoconjugates, and four types of glycans, glycosaminoglycans (GAGs), glycosphingolipid glycans (GSL), protein N- (NG) and O-glycans (OG), that collectively make up the bulk of the cellular glycome. While the biosynthetic pathways of these glycan types are distinct for the respective core glycan structure, the monosaccharide substrate building blocks and certain modifying enzymes are common between the synthesis of these glycan types. This suggests a potential interactivity between different glycoconjugate types, which we refer to as the “glycomic flux”, where glycosylation resources can be directed into other glycan pathways during a perturbation event, an area that has not been explored before. We have recently developed a Same Sample Sequential Multi-Glycomics (SSSMuG) workflow (Moh et. al. 2024) that sequentially releases all these glycan types from the same sample allowing for an in-depth, broad range glycomic analysis to study the glycomic flux.
Using this approach, we analysed the glycan types in HEK293F cells and their derived small extracellular vesicles (sEVs), where 5 specific glycan pathways were individually engineered through a “simple cell” strategy (Tian et. al. 2024). These HEK293F cells were cultured in a chemically defined, serum-free and protein-free media with minimal exogenous glycan contamination. In brief, our SSSMuG workflow identified a variety of glycomic flux responses that were distinct for each of the KO models. For example, in the N-glycan elongation targeting model (MGAT1 KO), apart from the expected termination of N-glycan synthesis at the Man5 oligomannose glycan, perturbations were also observed in the GAGs, GSL and OGs glycoprofiles. These observations show that the glycomic flux can result in a broad and diverse shift in the overall glycome by a single glycogene KO. With our new methodology, the glycome flux can now be characterized in detail.