Inhibiton of O-linked protein glycosylation
PUGNAc is a GlcNAc analog and potent inhibitor of O-GlcNAcase (OGA), which cleaves O-linked N-acetylglucosamine (β-O-linked 2-acetamido-2-deoxy-beta-D-glucopyranoside) from serine and threonine residues of cytoplasmic, nuclear and mitochondrial proteins (figure 1).
Figure 1. PUGNAc inhibits the removal of GlcNAc from glycosylated proteins.
Human isoform of the OGA enzyme (hOGA) is a 103-kDa protein with a N-terminal catalytic domain, a stalk domain and a pseudo-HAT C-terminal domain. The hOGA active site is composed of a conserved pair of aspartic acid residues (Asp174 and Asp175). Its catalytic domain has structural and mechanistic similarity to chitinases, N-acetylhexosaminidases and hyaluronidases from glycoside hydrolase families GH18, GH20, and GH56.
Inhibition of lysosomal degradation pathway of glycosphingolipids
PUGNAc inhibits also lysosomal β-hexosaminidases A and B. These two enzymes catalyse the removal of the terminal N-acetyl-D-hexosamine residue from globoside
. The A isoform is also involved in the synthesis of GM3 ganglioside
Figure 2. PUGNAc inhibits catalytic activity of lysosomal hexosaminidase isoenzymes A and B, involved in the synthesis of glycosphingolipids.
O-GlcNAcylation is a reversible post-translational protein modification involved in regulation of gene expression, proteasomal degradation, cell cycle and signalling. Protein O-glycosylation is a dynamic and reversible process, governed by O-GlcNAc cycling enzymes (figure 3) O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Dysregulation of O-linked protein glycosylation is associated with a series of pathologies, including cancer, diabetes and neurodegeneration. PUGNAc has been shown to increase global levels of O-GlcNAc and causes insulin resistance and to have protective effects on the post-traumatic cardiac function.
Figure 3. The level of O-GlcNAcylation is regulated by O-GlcNAc cycling enzymes.
Inhibition of hexosaminidases by PUGNAc has important implications in the study of lysosomal storage disorders. The inhibitor prevents hydrolysis of a terminal GalNAc
residue from glycosphingolipids and mimics GM2-gangliosidoses, which are inherited disorders with dysfunctional lysosomal hexosaminidases and neurodegenerative presentation. Globoside accumulation is the main cause of Sandhoff disease, and GM2
accumulation in Tay-Sachs disease.