O-GlcNAcase Activity Research Articles

Characterization of the O-GlcNAc protein modification in Xenopus laevis oocyte during oogenesis and progesterone-stimulated maturation

Little information exists about single N-acetylglucosamine modifications on proteins in growth and developmental model systems. To explore these phenomena, Xenopus laevis oocytes from stages I–VI of oogenesis were isolated and proteins analyzed on SDS-PAGE. The proteins were probed with antibodies specific for O-GlcNAc. Levels of the O-GlcNAc protein modification were highest in stages I and II, while decreasing in stages III–VI. The reduction in amount of O-GlcNAc-modified proteins was correlated to increases in apparent O-GlcNAcase (streptozotocin-inhibitable neutral hexosaminidase), activity involved in removing protein monoglycosylations. The O-GlcNAc modification was also characterized during progesterone-stimulated oocyte maturation. Although O-GlcNAcase activity appeared relatively constant between quiescent and matured stage VI oocytes, a small decrease in the levels of both total and specific O-GlcNAc-modified proteins was observed. Investigating the function of O-GlcNAc during maturation, oocytes were incubated with compounds known to modulate the levels of the O-GlcNAc protein modification and then stimulated to mature. Oocytes treated with compounds known to increase O-glycosylation consistently matured slower than non-treated controls, while oocytes treated with compounds that decrease O-glycosylation matured slightly faster than controls. The O-GlcNAc modification may play important roles in both the developmental and cell division processes of X. laevis oocytes.

Streptozotocin, an O-GlcNAcase inhibitor, blunts insulin and growth hormone secretion

Type 2 diabetes mellitus results from a complex interaction between nutritional excess and multiple genes. Whereas pancreatic β-cells normally respond to glucose challenge by rapid insulin release (first phase insulin secretion), there is a loss of this acute response in virtually all of the type 2 diabetes patients with significant fasting hyperglycemia. Our previous studies demonstrated that irreversible intracellular accumulation of a glucose metabolite, protein O-linked N-acetylglucosamine modification ( O-GlcNAc), is associated with pancreatic β-cell apoptosis. In the present study, we show that streptozotocin (STZ), a non-competitive chemical blocker of O-GlcNAcase, induces an insulin secretory defect in isolated rat islet cells. In contrast, transgenic mice with down-regulated glucose to glucosamine metabolism in β-cells exhibited an enhanced insulin secretion capacity. Interestingly, the STZ blockade of O-GlcNAcase activity is also associated with a growth hormone secretory defect and impairment of intracellular secretory vesicle trafficking. These results provide evidence for the roles of O-GlcNAc in the insulin secretion and possible involvement of O-GlcNAc in general glucose-regulated hormone secretion pathways.

Dynamic O-Glycosylation of Nuclear and Cytosolic Proteins: FURTHER CHARACTERIZATION OF THE NUCLEOCYTOPLASMIC β-N-ACETYLGLUCOSAMINIDASE, O-GlcNAcase

beta-O-linked N-acetylglucosamine (O-GlcNAc) is an abundant and dynamic post-translational modification implicated in protein regulation that appears to be functionally more similar to phosphorylation than to classical glycosylation. There are nucleocytoplasmic enzymes for the attachment and removal of O-GlcNAc. Here, we further characterize the recently cloned beta-N-acetylglucosaminidase, O-GlcNAcase. Both recombinant and purified endogenous O-GlcNAcase rapidly release free GlcNAc from O-GlcNAc-modified peptide substrates. The recombinant enzyme functions as a monomer and has kinetic parameters (K(m) = 1.1 mm for paranitrophenyl-GlcNAc, k(cat) = 1 s(-1)) that are similar to those of lysosomal hexosaminidases. The endogenous O-GlcNAcase appears to be in a complex with other proteins and is predominantly localized to the cytosol. Overexpression of the enzyme in living cells results in decreased O-GlcNAc modification of nucleocytoplasmic proteins. Finally, we show that the enzyme is a substrate for caspase-3 but, surprisingly, the cleavage has no effect on in vitro O-GlcNAcase activity. These studies support the identification of this protein as an O-GlcNAcase and identify important interactions and modifications that may regulate the enzyme and O-GlcNAc cycling.

Increased N-acetyl-β-glucosaminidase activity in primary breast carcinomas corresponds to a decrease in N-acetylglucosamine containing proteins

N-Acetylglucosamine (O-GlcNAc) modification on serine or threonine residues of cytoplasmic and nuclear proteins has become a more recognized intracellular covalent modification. Removal of this modification is carried out by N-acetyl-β-glucosaminidase (O-GlcNAcase). Since little information exists on monoglycosylation and O-GlcNAcase activity in mitogenic systems, we investigated O-GlcNAcase activity in primary breast tumors compared to matched normal adjacent breast tissue and examined enzymatic activity in relationship to the level of protein monoglycosylation. Using a variation of the acidic hexosaminidase activity assay, we demonstrated an increase in both O-GlcNAcase and lysosomal hexosaminidase activity in breast tumor tissue compared to matched adjacent tissue. Although no clear correlation with tumor grade or type was apparent among the samples examined (12 matched pairs), the increase in O-GlcNAcase and lysosomal hexosaminidase activity in tumor tissue was consistently elevated and statistically significant ( P<0.05). Protein monoglycosylation was evaluated using immunoblotting, affinity blotting, and radioactive labeling. While the variety of modified proteins was greater in tumor tissue compared to adjacent tissue, the total amount of O-GlcNAc monoglycosylation was significantly decreased in the tumor tissue especially on proteins in the molecular mass range of 45–65 kDa. O-GlcNAcase may be involved in the selective removal of O-GlcNAc on certain proteins in breast tumor tissue.

The potential mechanism of the diabetogenic action of streptozotocin: inhibition of pancreatic beta-cell O-GlcNAc-selective N-acetyl-beta-D-glucosaminidase.

Streptozotocin (STZ), an analogue of GlcNAc, inhibits purified rat spleen O-GlcNAc-selective N-acetyl-beta-D-glucosaminidase (O-GlcNAcase), the enzyme that removes O-GlcNAc from protein. We have shown previously that STZ increases pancreatic islet O-linked protein glycosylation. In light of these data, we investigated the possibility further that STZ causes beta-cell death by inhibiting O-GlcNAcase. In isolated islets, the time course and dose curve of STZ-induced O-glycosylation correlated with beta-cell toxicity. STZ inhibition of rat islet O-GlcNAcase activity also paralleled that of its beta-cell toxicity, with significant inhibition occurring at a concentration of 1 mM. In contrast, STZ inhibition of rat brain O-GlcNAcase and beta-TC3 insulinoma cell O-GlcNAcase was significantly right-shifted compared with islets, with STZ only significantly inhibiting activity at a concentration of 5 mM, the same concentration required for beta-TC3 cell toxicity. In comparison, N-methyl-N-nitrosourea, the nitric oxide-donating portion of STZ, did not cause increased islet O-glycosylation, beta-cell toxicity or inhibition of beta-cell O-GlcNAcase. Enhanced STZ sensitivity of islet O-GlcNAcase compared with O-GlcNAcase from other tissues or an insulinoma cell line suggests why actual islet beta-cells are particularly sensitive to STZ. Confirming this idea, STZ-induced islet beta-cell toxicity was completely blocked by GlcNAc, which also prevented STZ-induced O-GlcNAcase inhibition, but was not even partially blocked by glucose, glucosamine or GalNAc. Together, these data demonstrate that STZ's inhibition of beta-cell O-GlcNAcase is the mechanism that accounts for its diabetogenic toxicity.

The potential mechanism of the diabetogenic action of streptozotocin: inhibition of pancreatic β-cell O-GlcNAc-selective N-acetyl-β-d-glucosaminidase

Streptozotocin (STZ), an analogue of GlcNAc, inhibits purified rat spleen O-GlcNAc-selective N-acetyl-β-d-glucosaminidase (O-GlcNAcase), the enzyme that removes O-GlcNAc from protein. We have shown previously that STZ increases pancreatic islet O-linked protein glycosylation. In light of these data, we investigated the possibility further that STZ causes β-cell death by inhibiting O-GlcNAcase. In isolated islets, the time course and dose curve of STZ-induced O-glycosylation correlated with β-cell toxicity. STZ inhibition of rat islet O-GlcNAcase activity also paralleled that of its β-cell toxicity, with significant inhibition occurring at a concentration of 1mM. In contrast, STZ inhibition of rat brain O-GlcNAcase and β-TC3 insulinoma cell O-GlcNAcase was significantly right-shifted compared with islets, with STZ only significantly inhibiting activity at a concentration of 5mM, the same concentration required for β-TC3 cell toxicity. In comparison, N-methyl-N-nitrosourea, the nitric oxide-donating portion of STZ, did not cause increased islet O-glycosylation, β-cell toxicity or inhibition of β-cell O-GlcNAcase. Enhanced STZ sensitivity of islet O-GlcNAcase compared with O-GlcNAcase from other tissues or an insulinoma cell line suggests why actual islet β-cells are particularly sensitive to STZ. Confirming this idea, STZ-induced islet β-cell toxicity was completely blocked by GlcNAc, which also prevented STZ-induced O-GlcNAcase inhibition, but was not even partially blocked by glucose, glucosamine or GalNAc. Together, these data demonstrate that STZ's inhibition of β-cell O-GlcNAcase is the mechanism that accounts for its diabetogenic toxicity.

Streptozotocin-Induced β-Cell Death Is Independent of Its Inhibition of O-GlcNAcase in Pancreatic Min6 Cells

Streptozotocin (STZ) injection into experimental animals selectively causes massive β-cell death. The mechanism of this specific toxicity is not fully understood. Recently, it has been discovered that O-linked N-acetylglucosamine (O-GlcNAc) is enriched in the β-cells. It has been proposed that STZ toxicity may be due to its inhibition of neutral O-GlcNAcase activity, the enzyme that removes O-GlcNAc from cytosolic proteins (K. Liu et al., 2000, Proc. Natl. Acad. Sci. USA 97, 2820–2825). To further ascertain the role of O-GlcNAcase in β-cell death, we have used PUGNAc, a potent and specific O-GlcNAcase inhibitor, together with STZ in pancreatic Min6 cells. Both STZ and PUGNAc increased O-GlcNAc to similar levels on intracellular proteins. STZ, but not PUGNAc, decreased cellular protein synthesis by 66.0% within 8 h, killed 80.9% of the cells within 18 h, and decreased insulin secretion. STZ, but not PUGNAc, also caused genomic DNA fragmentation, suggesting that some of the cells were undergoing apoptosis. Prolonged treatment with PUGNAc (72 h) maintained high intracellular O-GlcNAc levels, but did not result in any apparent cell damage. Furthermore, the toxicity of STZ can be largely reversed by 3-aminobenzamide, a poly(ADP-ribose) polymerase inhibitor. These data strongly indicate that STZ-induced β-cell death is not caused by elevated intracellular O-GlcNAc levels, but instead likely involves poly(ADP-ribose) polymerase in the mechanism.