If you don’t look , you don’t see: Total glycome flux during cell differentiation

Recently, we published a new development of a holistic workflow for total glycome analysis of cells and tissues which we named the Same Sample Sequential Multi-Glycomics (SSSMuG) [Moh ESX, Dalal S, DeBono NJ, Kautto L, Wongtrakul-Kish K, Packer NH. SSSMuG: Same Sample Sequential Multi-Glycomics. Anal Chem. 2024.]. This workflow sequentially analyses 5 glycan types (polysialic acid, GAGs, GSL glycans, protein N- and O-glycans) of the overall glycome, sequentially from a single sample.

We now have data (manuscript in preparation) from a HEK293F cell line model, where perturbation (by genetic knockouts) of different glycosylation pathway enzymes, results in unpredicted changes of the products of other glycosylation pathways in the extracellular vesicles. This inter-dependency of glycosylation pathways has not been considered previously and can be thought of as the “glycome flux”, where glycan resources are channeled into various pathways based on cellular supply and demand.

In this application, to investigate the extent of the glycomics flux that occurs in a natural system rather than with a gene knockout, we will explore two in vitro cell differentiation models that do not require any genetic manipulations for the glyco-metabolic load to shift;
1. a bronchial epithelial cell line (Calu-3) that secretes mucus (increased mucin O-glycan load) when the cells differentiate upon exposure to the air-liquid interface, and
2. a mouse fibroblast cell line (3T3-L1 ) that produces glycosaminoglycans (increased GAG load) when differentiating into adipocytes.

By analyzing the total glycome of the cells in these two natural differentiation models, we aim to understand the extent of the glycomics flux over biologically relevant processes.