A major role for the intracellular posttranslational modification O-GlcNAc appears to be the inhibition of protein aggregation. Most of the previous studies in this area have focused on O-GlcNAc modification of the amyloid-forming proteins themselves. Here, we use synthetic protein chemistry to discover that O-GlcNAc also activates the anti-amyloid activity of certain small heat shock proteins (sHSPs), a potentially more important modification event that can act broadly and substoichiometrically. More specifically, we find that O-GlcNAc increases the ability of sHSPs to block the amyloid formation of both α-synuclein and Aβ(1-42). Mechanistically, we show that O-GlcNAc near the sHSP IXI-domain prevents its ability to intramolecularly compete with substrate binding. Finally, we find that although O-GlcNAc levels are globally reduced in Alzheimer’s disease brains, the modification of relevant sHSPs is either maintained or increased, suggesting a mechanism to maintain these potentially protective O-GlcNAc modifications. Our results have important implications for neurodegenerative diseases associated with amyloid formation and potentially other areas of sHSP biology.

This review details a brief history of the synthesis and characterization of metabolic chemical reporters used to study glycosylation before describing recent applications and finishing with considerations and limitations of reporter molecules.

The amounts of the intracellular glycosylation, O-GlcNAc modification, are increased in essentially all tumors when compared to healthy tissue, and lowering O-GlcNAcylation levels results in reduced tumorigenesis and increased cancer cell death. Therefore, the pharmacological reduction of O-GlcNAc may represent a therapeutic vulnerability. The most direct approach to this goal is the inhibition of O-GlcNAc transferase (OGT), the enzyme that directly adds the modification to proteins. However, despite some recent success, this enzyme has proven difficult to inhibit. An alternative strategy involves starving OGT of its sugar substrate UDP-GlcNAc by targeting enzymes of the hexosamine biosynthetic pathway (HBP). Here, we explore the potential of the rate-determining enzyme of this pathway, glutamine fructose-6-phosphate amidotransferase (GFAT). We first show that CRISPR-mediated knockout of GFAT results in inhibition of cancer cell growth in vitro and a xenograft model that correlates with O-GlcNAcylation levels. We then demonstrate that pharmacological inhibition of GFAT sensitizes a small panel of cancer cells to undergo apoptosis in response to diamide-induced oxidative stress. Finally, we find that GFAT expression and O-GlcNAc levels are increased in a spontaneous mouse model of liver cancer. Together these experiments support the further development of inhibitors of the HBP as an indirect approach to lowering O-GlcNAcylation levels in cancer.

Many intracellular proteins are modified by N-acetylglucosamine, a posttranslational modification termed O-GlcNAc. This modification is found on serine and threonine side-chains and has the potential to regulate signaling pathways through interplay with phosphorylation. Here, we discover and characterize one such example. We find that O-GlcNAc levels control the sensitivity of fibroblasts to actin contraction induced by the signaling lipid sphingosine-1-phosphate (S1P), culminating in the phosphorylation of myosin light chain (MLC) and cellular contraction. Specifically, O-GlcNAc modification of the phosphatase subunit MYPT1 inhibits this pathway by blocking MYPT1 phosphorylation, maintaining its activity and causing the dephosphorylation of MLC. Finally, we demonstrate that O-GlcNAc levels alter the sensitivity of primary human dermal fibroblasts in a collagen-matrix model of wound healing. Our findings have important implications for the role of O-GlcNAc in fibroblast motility and differentiation, particularly in diabetic wound healing.

Glycan binding often mediates extracellular macromolecular recognition events. Accurate characterization of these binding interactions can be difficult because of dissociation and scrambling that occur during purification and analysis steps. Use of photocrosslinking methods has been pursued to covalently capture glycan-dependent interactions in situ; however, use of metabolic glycan engineering methods to incorporate photocrosslinking sugar analogs is limited to certain cell types. Here, we report an exoenzymatic labeling method to add a diazirine-modified sialic acid (SiaDAz) to cell surface glycoconjugates. The method involves the chemoenzymatic synthesis of diazirine-modified CMP-sialic acid (CMP-SiaDAz), followed by sialyltransferase-catalyzed addition of SiaDAz to desialylated cell surfaces. Cell surface SiaDAzylation is compatible with multiple cell types and is facilitated by endogenous extracellular sialyltransferase activity present in Daudi B cells. This method for extracellular addition of α2-6-linked SiaDAz enables UV-induced crosslinking of CD22, demonstrating the utility for covalent capture of glycan-mediated binding interactions.