GlcNAcstatins are nanomolar inhibitors of human O-GlcNAcase inducing cellular hyper-O-GlcNAcylation.
Bottom Line: Mutagenesis studies in a bacterial OGA, guided by the structure of a GlcNAcstatin complex, provides insight into the role of conserved residues in the human OGA active site.GlcNAcstatins are cell-permeant and, at low nanomolar concentrations, effectively modulate intracellular O-GlcNAc levels through inhibition of OGA, in a range of human cell lines.Thus these compounds are potent selective tools to study the cell biology of O-GlcNAc.
Affiliation: University of Dundee, Scotland, UK.
O-GlcNAcylation is an essential, dynamic and inducible post-translational glycosylation of cytosolic proteins in metazoa and can show interplay with protein phosphorylation. Inhibition of OGA (O-GlcNAcase), the enzyme that removes O-GlcNAc from O-GlcNAcylated proteins, is a useful strategy to probe the role of this modification in a range of cellular processes. In the present study, we report the rational design and evaluation of GlcNAcstatins, a family of potent, competitive and selective inhibitors of human OGA. Kinetic experiments with recombinant human OGA reveal that the GlcNAcstatins are the most potent human OGA inhibitors reported to date, inhibiting the enzyme in the sub-nanomolar to nanomolar range. Modification of the GlcNAcstatin N-acetyl group leads to up to 160-fold selectivity against the human lysosomal hexosaminidases which employ a similar substrate-assisted catalytic mechanism. Mutagenesis studies in a bacterial OGA, guided by the structure of a GlcNAcstatin complex, provides insight into the role of conserved residues in the human OGA active site. GlcNAcstatins are cell-permeant and, at low nanomolar concentrations, effectively modulate intracellular O-GlcNAc levels through inhibition of OGA, in a range of human cell lines. Thus these compounds are potent selective tools to study the cell biology of O-GlcNAc.
Mentions: The intended application of the GlcNAcstatins is to inhibit hOGA in live human cells, resulting in hyper-O-GlcNAcylation by disrupting the balance between O-GlcNAc transfer and hydrolysis. Such modulation of O-GlcNAc levels would allow for the study of O-GlcNAc-dependent signal transduction processes. To evaluate the use of the GlcNAcstatins for cell biological studies, HEK-293 cells were exposed to various concentrations of GlcNAcstatins for 6 h, followed by investigation of O-GlcNAc levels on cellular proteins by Western blot analysis using an anti-O-GlcNAc antibody (CTD110.6) (Figure 3A). GlcNAcstatins B–D increase cellular O-GlcNAc levels of numerous intracellular proteins when used at concentrations as low as 20 nM. GlcNAcstatins A and E appear to be less potent as quantitatively assessed from the Western blots, requiring micromolar concentrations in the cell-based assay for a marked effect inside the cells. For GlcNAcstatin E, this is in agreement with the in vitro inhibition data that show that this compound is the weakest hOGA inhibitor (Table 1). The reduced cellular activity of GlcNAcstatin A could be due to differences in membrane permeability resulting from the less hydrophobic nature of the C-2 carboxymethyl substituent. Taken together, these results suggest that GlcNAcstatins are cell-permeant compounds that modulate O-GlcNAcylation levels within the cells by inhibiting hOGA.