Su-Il Do

Denitrosylation of S-nitrosylated OGT is triggered in LPS-stimulated innate immune response

O-linked N-acetylglucosaminyltransferase (OGT)-mediated protein O-GlcNAcylation has been revealing various aspects of functional significance in biological processes, such as cellular signaling and activation of immune system. We found that OGT is maintained as S-nitrosylated form in resting cells, and its denitrosylation is triggered in innate immune response of lipopolysaccharide (LPS)-treated macrophage cells. S-nitrosylation of OGT strongly inhibits its catalytic activity up to more than 80% of native OGT, and denitrosylation of OGT leads to protein hyper-O-GlcNAcylation. Furthermore, blockage of increased protein O-GlcNAcylation results in significant loss of nitric oxide and cytokine production. We propose that denitrosylation of S-nitrosylated OGT is a direct mechanism for upregulation of OGT activity by which immune defense is critically controlled in LPS-stimulated innate immune response.

Sp1 modulates ncOGT activity to alter target recognition and enhanced thermotolerance in E. coli.

cDNAs encoding three isoforms of OGT (ncOGT, mOGT, and sOGT) were expressed in Escherichia coli in which the coexpression system of OGT with target substrates was established in vivo. No endogenous bacterial proteins were significantly O-GlcNAcylated by any type of OGT isoform while co-expressed p62 and Sp1 were strongly O-GlcNAcylated by ncOGT. These results suggest that most of bacterial proteins appear not to be recognized as right substrates by mammalian OGT whereas cytosolic environments may supply UDP-GlcNAc enough to proceed to O-GlcNAcylation in E. coli. Under these conditions, sOGT was auto-O-GlcNAcylated whereas ncOGT and mOGT were not. Importantly, we found that when Sp1 was coexpressed, ncOGT can O-GlcNAcylate not only Sp1 but also many bacterial proteins. Our findings suggest that Sp1 may modulate the capability of target recognition of ncOGT by which ncOGT can be led to newly recognize bacterial proteins as target substrates, finally generating the O-glyco-bacteria. Our results demonstrate that the O-glyco-bacteria showed enhanced thermal resistance to allow cell survival at a temperature as high as 52 degrees C.

Pathophysiological implication of ganglioside GM3 in early mouse embryonic development through apoptosis.

Apoptosis may occur in early embryos where the execution of essential developmental events has failed, and gangliosides, sialic acid-conjugated glycosphingolipids, are proposed to regulate cell differentiation and growth. To evaluate the regulatory roles of ganglioside GM3 in early embryonic development, this study examined its expressional patterns in apoptotic cells during early embryonic development in mice. Pre-implanted embryos were obtained byin vitro fertilization, which were treated at the 4-cell stage with three the apoptosis inducers, actinomycin D, camptothecin and cycloheximide, for 15 h. All three inducers significantly increased the percentage of apoptotic cells, as measured using a TUNEL method, but remarkably reduced the total cell numbers. The numbers of morula and blastocyst stages were significantly decreased by treatment of the embryos with the three apoptosis inducers compared with the control, with a similar result also observed in the number of blastomeres. Staining of early embryos with Hoechst 33342 revealed a significant percentage of apoptotic nuclei. Prominent immunofluorescence microscopy revealed a significant difference in the ganglioside GM3 expression in apoptotic embryos compared with the control, and RT-PCR also demonstrated a dramatic increase in ganglioside GM3 synthase mRNA in the apoptotic embryos. These results suggest that ganglioside GM3 may be pathophysiologically implicated in the regulation of early embryonic development through an apoptotic mechanism.

Aβ-affected pathogenic induction of S-nitrosylation of OGT and identification of Cys-NO linkage triplet.

Mechanistic link of protein hypo-O-GlcNAcylation to the pathogenesis of Alzheimers disease (AD) remains unclear. Here, we found that S-nitrosylation of O-linked N-acetylglucosaminyltransferase (SNO-OGT) was induced by β-amyloid peptide (Aβ) exposure to SK-N-MC and SK-N-SH human neuroblastoma cells. Subsequently, Aβ-induced SNO-OGT led to protein hypo-O-GlcNAcylation globally including tau hypo-O-GlcNAcylation. Our results support that underlying mechanism for induction of SNO-OGT comprises the concerted action of Aβ-triggered Ca2+ entry into cells and nNOS-catalyzed NO production. Intriguingly, OGT was found to be associated with nNOS and its association was enhanced during Aβ treatment. In parallel with SNO-OGT-mediated tau hypo-O-GlcNAcylation, Aβ led to SNO-Akt-mediated GSK3β activation for tau phosphorylation, suggesting that tau hyperphosphorylation is established by synergistic connection between SNO-OGT and GSK3β activation. We also observed that Aβ-neurotoxicity including both reactive oxygen species (ROS) production and cell death was amplified with DON treatment, whereas it was restored by PUGNAc treatment, GlcNH2 treatment or OGT overexpression. Early time-course Aβ-monitoring assay revealed that premaintained hyper-O-GlcNAcylation inside cells blocked not only Aβ-triggered Ca2+ entry into cells but also induction of SNO-OGT and SNO-Akt. Together, these findings suggest that induction of SNO-OGT by Aβ exposure is a pathogenic mechanism to cause cellular hypo-O-GlcNAcylation by which Aβ neurotoxicity is executed, and conversely, hyper-O-GlcNAcylation within cells can defend against Aβ neurotoxicity. Furthermore, our Cys mapping demonstrates that cysteine-nitric oxide (Cys-NO) linkages in SNO-OGT occur at triple Cys845, Cys921, and Cys965 residues in C-terminal catalytic domain (C-CAT), suggesting that Cys-NO linkage triplet in SNO-OGT is associated with null OGT activity.

Valproic acid-mediated transcriptional regulation of human GM3 synthase (hST3Gal V) in SK-N-BE(2)-C human neuroblastoma cells

AbstractAim:To investigate whether valproic acid (VPA) modulates human GM3 synthase (hST3Gal V) mRNA expression, as a part of ganglioside GM3 biosynthesis, in human neuroblastoma cells.Methods:Using RT-PCR and immunofluo-rescent confocal microscopy, we examined hST3Gal V mRNA and GM3 levels during VPA-induced differentiation of human neuroblastoma SK-N-BE(2)-C cells. We characterized the VPA-inducible promoter region within the hST3-Gal V gene using luciferase constructs carrying 5′-deletions of the hST3Gal V promoter.Results:RT-PCR indicated that VPA-mediated hST3Gal V induction is transcriptionally regulated. Functional analysis of the 5′-flanking region of the hST3Gal V gene demonstrated that the -177 to -83 region, which contains a cAMP-responsive element (CRE) at -143, functions as the VPA-inducible promoter by actively binding CRE binding protein (CREB). In addition, site-directed mutagenesis and electrophoretic mobility shift assay indicated that the CRE at -143 is crucial for the VPA-induced expression of hST3Gal V in SK-N-BE(2)-C cells.Conclusion:Our results isolated the core promoter region in the hST3Gal V promoter, a CRE at -143, and demonstrated that it is essential for transcriptional activation of hST3Gal V in VPA-induced SK-N-BE(2)-C cells. Subsequent CREB binding to this CRE mediates VPA-dependent upregulation of hST3Gal V gene expression.

Generation of novel chimeric LacdiNAcS by gene fusion of α-lactalbumin and β1,4-galactosyltransferase 1

Novel chimeric lacdiNAc (GalNAc(β1-4)GlcNAc) synthase (c-LacdiNAcS) was generated by gene fusion of α-lactalbumin (α-LA) and β1,4-galactosyltransferase 1 (β1,4-GalT1). c-LacdiNAcS was expressed in Lec8 Chinese hamster ovary (Lec8 CHO) cells and exhibited N-acetylgalactosaminyltransferase (GalNAcT) activity in the absence of exogenous α-LA as well as other glycosyltransferase activities including lactose synthase (LacS), and β1,4-GalT. These glycosyltransferase activities of c-LacdiNAcS were compared to those activities induced in LacS system under the co-presence of bovine β1,4-GalT1 and α-LA, indicating that each domain of α-LA and β1,4-GalT1 on c-LacdiNAcS is not only folding correctly, but also interacting together. Furthermore, c-LacdiNAcS was found to be auto-lacdiNAcylated and can synthesize lacdiNAc structures on cellular glycoproteins, demonstrating that GalNAcT activity of c-LacdiNAcS is functional in Lec8 CHO cells.