Hyperglycemia and the O-GlcNAc transferase in rat aortic smooth muscle cells: elevated expression and altered patterns of O-GlcNAcylation.

Hyperglycemia leads to vascular disease specific to diabetes mellitus. This pathology, which results from abnormal proliferation of smooth muscle cells in arterial walls, may lead to cataract, renal failure, and atherosclerosis. The hexosamine biosynthetic pathway is exquisitely responsive to glucose concentration and plays an important role in glucose-induced insulin resistance. UDP-GlcNAc: polypeptide O-N-acetylglucosaminyltransferase (O-GlcNAc transferase; OGTase) catalyzes the O-linked attachment of single GlcNAc moieties to serine and threonine residues on many cytosolic or nuclear proteins. Polyclonal antibody against OGTase was used to examine the expression of OGTase in rat aorta and aortic smooth muscle (RASM) cells. OGTase enzymatic activity and expression at the mRNA and protein levels were determined in RASM cells cultured at normal (5 mM) and at high (20 mM) glucose concentrations. OGTase mRNA and protein are expressed in both endothelial cells and smooth muscle cells in the aorta of normal rats. In both cell types, the nucleus is intensely stained, while the cytoplasm stains diffusely. Immunoelectron microscopy shows that OGTase is localized to euchromatin and around the myofilaments of smooth muscle cells. In RASM cells grown in 5 mM glucose, OGTase is also located mainly in the nucleus. Hyperglycemic RASM cells also display a relative increase in OGTase's p78 subunit and an overall increase protein and activity for OGTase. Biochemical analyses show that hyperglycemia qualitatively and quantitatively alters the glycosylation or expression of many O-GlcNAc-modified proteins in the nucleus. These results suggest that the abnormal O-GlcNAc modification of intracellular proteins may be involved in glucose toxicity to vascular tissues.

Increased O-GlcNAc transferase in pancreas of rats with streptozotocin-induced diabetes.

AIMS/HYPOTHESIS: Streptozotocin (STZ), a chemically reactive analogue of N-acetylglucosamine, induces necrosis of the beta cells, resulting in diabetes mellitus. Glucose-induced insulin resistance is mediated by increased activity of the hexosamine pathway. We aimed to examine the regulation of O-GlcNAc transferase expression and activity in the normal and streptozotocin diabetic pancreas. METHODS: Rats were made diabetic by an injection of streptozotocin (65 mg/kg). The expression of O-GlcNAc transferase protein was examined by immunoblot analysis. Activity of O-GlcNAc transferase was assayed by the incorporation of [3H]GlcNAc into the synthetic peptide. Localization of O-GlcNAc transferase was done by immunohistochemistry. The change of O-GlcNAc modification of proteins was examined by immunoblot analysis. RESULTS: In the STZ-induced diabetic pancreas, a severe loss of beta cells was observed, whereas alpha cells had increased in number. The diabetic pancreas showed an increase in the expression of O-GlcNAc transferase at the protein level and the O-GlcNAc transferase activity in it was increased significantly (p < 0.05). An increase in the immunostaining intensity in the cytoplasm of islet beta cells was also observed in the diabetic pancreas, whereas exocrine cells and islet cells other than beta cells showed little change in immunostaining intensity. The pancreas of STZ-diabetic rats showed a 3.1-fold increase in total cellular O-GlcNAc-modified proteins. CONCLUSION/INTERPRETATION: These findings indicate that O-GlcNAc transferase plays an important part in the modulation of O-GlcNAc concentrations in the pancreas and suggest that the increase in O-GlcNAc modification of the proteins correlates closely with diabetes.

Localization of the O-linked N-acetylglucosamine transferase in rat pancreas.

O-linked N-acetylglucosamine transferase (OGT) catalyzes the attachment ofN-acetylglucosamine (GlcNAc) monosaccharides to the hydroxyl group of serine or threonine residues of intracellular proteins and may play an important role in the hexosamine pathway. Glucose-induced insulin resistance is mediated by increased activity of the hexosamine pathway. In the present study, we examined the localization of OGT mRNA and OGT protein in the rat pancreas. The sites of OGT mRNA expression were determined by in situ hybridization histochemistry with a digoxigenin (DIG)-labeled antisense cRNA probe. Intense hybridization signals were present in the exocrine acinar cells, while weaker ones were detected in the islets of Langerhans. This distribution was confirmed using additional antisense cRNA or oligo-cDNA probes complementary to different regions of OGT mRNA. In addition, immunofluorescence staining with antibody raised against OGT stained both the exocrine acinar cells and endocrine islet cells. In the acinar cell nucleus, the zymogen granule region and contour of the cell were intensely stained. In the islets of Langerhans, especially in the alpha-cells, intense staining with anti-OGT antibody was observed. These staining patterns were almost identical to those seen when staining for the O-linked GlcNAc (O-GlcNAc) modification. Immuno-electron microscopy showed that OGT is localized to the euchromatin of the nucleus and around the secretory granules of exocrine acinar cells and endocrine islet cells. These results suggest that OGT is involved in the regulation of transcription and of granular secretion. Thus, one or more O-GlcNAcylated proteins may be important components of the glucose-sensing mechanism in the pancreas.

Regulation of a cytosolic and nuclear O-GlcNAc transferase. Role of the tetratricopeptide repeats.

The O-GlcNAc transferase (OGT) is a unique nuclear and cytosolic glycosyltransferase that contains multiple tetratricopeptide repeats. We have begun to characterize the mechanisms regulating OGT using a combination of deletion analysis and kinetic studies. Here we show that the p110 subunit of the enzyme forms both homo- and heterotrimers that appear to have different binding affinities for UDP-GlcNAc. The multimerization domain of OGT lies within the tetratricopeptide repeat domain and is not necessary for activity. Kinetic analyses of the full-length trimer and the truncated monomer forms of OGT suggest that both forms function through a random bi-bi kinetic mechanism. Both the monomer and trimer have similar specific activities and similar K(m) values for peptide substrates. However, they differ in their binding affinities for UDP-GlcNAc, indicating that subunit interactions affect enzyme activity. The findings that recombinant OGT has three distinct K(m) values for UDP-GlcNAc and that UDP-GlcNAc concentrations modulates the affinity of OGT for peptides suggest that OGT is exquisitely regulated by the levels of UDP-GlcNAc within the nucleus and cytoplasm.

Dynamic glycosylation of nuclear and cytosolic proteins. Cloning and characterization of a unique O-GlcNAc transferase with multiple tetratricopeptide repeats.

O-Linked N-acetylglucosamine (O-GlcNAc) glycosylation is a dynamic modification of eukaryotic nuclear and cytosolic proteins analogous to protein phosphorylation. We have cloned and characterized a novel gene for an O-GlcNAc transferase (OGT) that shares no sequence homology or structural similarities with other glycosyltransferases. The OGT gene is highly conserved (up to 80% identity) in all eukaryotes examined. Unlike previously described glycosyltransferases, OGT is localized to the cytosol and nucleus. The OGT protein contains multiple tandem repeats of the tetratricopeptide repeat motif. The presence of tetratricopeptide repeats, which can mediate protein-protein interactions, suggests that OGT may be regulated by protein interactions that are independent of the enzyme's catalytic site. The OGT is also modified by tyrosine phosphorylation, indicating that tyrosine kinase signal transduction cascades may play a role in modulating OGT activity.

Purine biosynthetic genes are required for cadmium tolerance in Schizosaccharomyces pombe.

Phytochelatins (PCs) are metal-chelating peptides produced in plants and some fungi in response to heavy metal exposure. A Cd-sensitive mutant of the fission yeast Schizosaccharomyces pombe, defective in production of a PC-Cd-sulfide complex essential for metal tolerance, was found to harbor mutations in specific genes of the purine biosynthetic pathway. Genetic analysis of the link between metal complex accumulation and purine biosynthesis enzymes revealed that genetic lesions blocking two segments of the pathway, before and after the IMP branchpoint, are required to produce the Cd-sensitive phenotype. The biochemical functions of these two segments of the pathway are similar, and a model based on the alternate use of a sulfur analog substrate is presented. The novel participation of purine biosynthesis enzymes in the conversion of the PC-Cd complex to the PC-Cd-sulfide complex in the fission yeast raises an intriguing possibility that these same enzymes might have a role in sulfur metabolism in the fission yeast S. pombe, and perhaps in other biological systems.

Heavy metal tolerance in the fission yeast requires an ATP-binding cassette-type vacuolar membrane transporter.

In response to heavy metal stress, plants and certain fungi, such as the fission yeast Schizosaccharomyces pombe, synthesize small metal-binding peptides known as phytochelatins. We have identified a cadmium sensitive S. pombe mutant deficient in the accumulation of a sulfide-containing phytochelatin-cadmium complex, and have isolated the gene, designated hmt1, that complements this mutant. The deduced protein sequence of the hmt1 gene product shares sequence identity with the family of ABC (ATP-binding cassette)-type transport proteins which includes the mammalian P-glycoproteins and CFTR, suggesting that the encoded product is an integral membrane protein. Analysis of fractionated fission yeast cell components indicates that the HMT1 polypeptide is associated with the vacuolar membrane. Additionally, fission yeast strains harboring an hmt1-expressing multicopy plasmid exhibit enhanced metal tolerance along with a higher intracellular level of cadmium, implying a relationship between HMT1 mediated transport and compartmentalization of heavy metals. This suggests that tissue-specific overproduction of a functional hmt1 product in transgenic plants might be a means to alter the tissue localization of these elements, such as for sequestering heavy metals away from consumable parts of crop plants.