Browsing by Subject "glycobiology"

Glycosylation is a universally conserved and ubiquitous posttranslational modification required for myriad cellular processes. Biosynthesis of these moieties, termed glycans, relies on nucleotide-sugars (NS), abundant metabolites that serve as monosaccharide donors for glycosyltransferases. In vivo, signal-dependent fluctuations in NS levels are required to maintain normal cell physiology and are dysregulated in disease, but how mammalian cells regulate NS levels and pathway flux remains largely uncharacterized. To address this knowledge gap, we examined uridine diphosphate (UDP)-galactose 4’-epimerase (GALE), which interconverts two pairs of essential NSs. In early work, we discovered that GALE knockdown sensitizes human cells to ER stress, suggesting that GALE is important for stress responses. While increased NS levels during ER stress have been reported previously, we found that GALE knockdown blunted this response.

GALE deletion via CRISPR/Cas9 in human cells triggered major imbalances in its substrate NSs along with changes in fatty acid levels, suggesting that GALE is crucial for whole-cell metabolism. The novel link between GALE and ostensibly unrelated metabolic pathways may prove important for understanding disorders of galactose metabolism. Altered NS levels led to dramatic changes in glycolipids and glycoproteins, including a subset of integrins and the Fas death receptor. These changes in glycan biosynthesis directly impacted protein function, as GALE-/- cells exhibit hypersensitivity to Fas ligand (FasL)-induced apoptosis. Critically, monosaccharide supplementation reverses both Fas misglycosylation and FasL sensitivity, supporting a glycan-centric mechanism for this phenotype. Our results reveal a new role for GALE-mediated NS regulation in supporting death receptor signaling and may have implications for the molecular etiology of illnesses characterized by NS imbalances, including galactosemia and metabolic syndrome.

To probe effects of integrin misglycosylation in GALE-/- cells, we leveraged a quantitative assay for cell adhesion, finding that GALE-/- cells adhered more robustly than controls on a variety of extracellular matrices. Subsequent experiments revealed technical limitations of this assay and the link between NS metabolism, integrin glycosylation, and cell adhesion remains inconclusive. We observed GALE-dependent changes in integrin stability or lifetime, suggesting that glycosylation has important functional consequences for integrin subunits.

In a model of B cell maturation, we found that GALE protein levels are increased by B cell stimulation. Since B cell stimulation triggers the transcription factor Xbp1s, of which GALE is a reported target, this suggested an exciting connection between B cell maturation and NS metabolism. However, subsequent studies using Xbp1-/- B cells and drugs preventing Xbp1 activation suggest that GALE upregulation is independent of Xbp1s in this context.

Intracellular glycosylation via O-linked N-acetylglucosamine (O-GlcNAc) is a rapidly reversible signaling moiety in animals. In response to ER stress, increased O-GlcNAc glycosylation promotes cell survival in multiple contexts, but specific substrate proteins modified during this response remain unknown. To discover ER stress-regulated glycoproteins and characterize the functional impact of their glycosylation, we employed a chemical biology glycoproteomics workflow combining an azide-functionalized hexosamine mimetic with SILAC proteomics. Our results demonstrate that a subset of glycoproteins is differentially glycosylated in response to the ER-stress inducing drug tunicamycin, suggesting a regulated rearrangement of the intracellular glycome.

Although the structural and enzymological characteristics of NS biosynthetic enzymes are well-studied, the consequences of altered NS metabolism in response to signals or disease states is understudied. In this work, we used both genetic models of NS imbalance and stimuli previously reported to affect NS metabolism to measure downstream glycosylation and consequences thereof. This work lays the foundation for a more comprehensive understanding of NS metabolic regulation in mammalian physiology.