GlcNAcstatins are nanomolar inhibitors of human O-GlcNAcase inducing cellular hyper-O-GlcNAcylation

Abstract

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.

Figure 1. Kinetic characterization of GlcNAcstatin derivatives

(A) Chemical structures of the glycosidase inhibitors PUGNAc, nagstatin and the GlcNAcstatin scaffold. (B) Lineweaver–Burk analysis of hOGA steady-state kinetics measured in the presence of 0–40 nM GlcNAcstatin C. Data were fitted using the standard equation for competitive inhibition in the GraFit program (Erithacus Software). (C) Dose–response curve of GlcNAcstatins A–E incubated with lysosomal HexA/HexB. Data were fitted using the standard IC₅₀ equation in the GraphFit program (Erithacus Software).

Figure 2. Structural analysis of GlcNAcstatin derivatives and PUGNAc in complex with CpOGA

(A) Stereo view of GlcNAcstatin D (sticks with green carbon, red oxygen and blue nitrogen atoms) in the active site of CpOGA (sticks with grey carbon). Hydrogen bonds are indicated by black broken lines. Unbiased |F_o|–|F_c|, ϕ_calc electron density map (2.75 σ) is shown as a cyan chickenwire. (B) Stereo view of superimposed crystallographically determined complexes of CpOGA with GlcNAcstatin C (PDB number 2J62) (colour scheme as in A) and PUGNAc (PDB number 2CBJ) (sticks with light blue carbon), black broken lines showing hydrogen bonds for the CpOGA–GlcNAcstatin complex.

Figure 3. Immunoblot detection of O-GlcNAc modifications on cellular proteins using an anti-O-GlcNAc antibody

The increase in O-GlcNAc levels in comparison with untreated samples is shown in the histogram underneath the blot. (A) HEK-293 cells were treated with GlcNAcstatins A–E for 6 h with the concentrations indicated. (B) GlcNAcstatin C was added to HeLa, HT-1080, SH-SY5Y or U-2 OS cells for 6 h with the identical inhibitor concentrations as in (A). The molecular mass in kDa is indicated on the left-hand side of each blot.