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

doi:10.1016/j.bbrc.2011.03.115

Biochemical and Biophysical Research Communications

Volume 408, Issue 1, 29 April 2011, Pages 52-57

https://doi.org/10.1016/j.bbrc.2011.03.115 Get rights and content

Abstract

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.

Highlights

► S-nitrosylated OGT (SNO-OGT) in resting state is denitrosylated upon LPS stimulation. ► Denitrosylation of OGT leads to protein hyper-O-GlcNAcylation. ► Attenuation of hyper-O-GlcNAcylation results in abnormality of NF_kB activity. ► Protein hyper-O-GlcNAcylation critically functions in NO and cytokine production. ► Denitrosylation of SNO-OGT is a key process for innate immune regulation.

Introduction

Since O-GlcNAc was discovered [1], it has been known that Ser/Thr-O-linked glycosylation (O-GlcNAcylation) on a variety of cytosolic and nuclear proteins is catalyzed by O-linked N-acetylglucosaminyltransferase (OGT) with addition of monosaccharide residue of N-acetylglucosamine (GlcNAc) [2], [3]. Molecular basis of substrate recognition of OGT and catalysis by OGT has been revealed [4], [5], and OGT-catalyzed protein O-GlcNAcylation is widely involved in distinct regulation of cellular signaling [6], hyperthermal stress [7], diseases [8], [9], and immune systems [10], [11], [12]. OGT gene has been cloned in a broad range of species [13], [14], [15], [16], [17] and posttranslational modification (PTM) of OGT polypeptide has been reported as two types, such as O-GlcNAc modification and tyrosine phosphorylation [13]. Little has been known about influential potency of PTM on OGT catalytic activity and so far, no other type of PTM in OGT protein backbone has been identified.

Innate immune signaling is initiated by specific interaction of pathogen ligands with Toll-like receptors (TLRs) and proinflammatory signaling for antipathogenic defense is triggered to induce nitric oxide (NO) and cytokine production [18], [19]. It has been reported that NO, a principle bactericidal mediator is produced primarily by inducible NO synthase (iNOS) under lipopolysaccharide (LPS) stimulation in macrophage cells [20], [21]. Moreover, it has been suggested that protein S-nitrosylation of cysteine thiols in NO-generating cells could be an important regulator in intracellular signaling [22], [23].

In this report, to gain insight into defensive roles of O-GlcNAcylation in innate immune stress, we explored the protein S-nitrosylation that can control OGT activity in lipopolysaccharide (LPS)-stimulated macrophage cells. Here, we provide the first evidence that OGT was S-nitrosylated in resting state and denitrosylation of S-nitrosylated OGT (SNO-OGT) is triggered upon LPS stimulation both in vitro and in vivo. Our results show that S-nitrosylation together with denitrosylation is previously unrecognized PTM that directly regulates OGT activity in innate immune system.

Section snippets

Materials

Antibodies for NF_kB p65 (F-6), iNOS (N-20), NOS3 (C-20), actin, PCNA, MBP-tag (N-17), His₆-tag (G-18), and Protein A/G-agarose (PLUS) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies for O-linked GlcNAc (RL2) and Streptavidin-HRP were obtained from Abcam (USA) and Vector (USA), respectively. Greiss reagents, Nitrate/Nitrite Assay kit Colorimetric, DON (6-diazo-5-oxo-L-norleucine), OGT antibody (DM17), LPS (Echerichia coli 0111), GSNO (S-nitroso-glutathione), p

Enhanced protein O-GlcNAcylation in LPS-treated macrophage cells in vitro and in vivo

To investigate whether cellular level of protein O-GlcNAcylation was changed during in vitro LPS stimulation of RAW264.7 cells, we analyzed O-GlcNAcylation by RL2 immunoblotting. Protein O-GlcNAcylation was initiated to be increased at early phase of 2 h, maximized at 12 h LPS treatment, and this enhanced O-GlcNAcylation was prolonged until 24 h LPS treatment whereas endogenous OGT level was not affected (Fig. 1A). Expression pattern of inducible NO synthase (iNOS) was consistently observed with

Acknowledgments

We thank Dr. Ki-Young Lee (GG Pharmaceutical Institute) for helpful discussion and critical reading of the manuscript; Il-Soo Shin (Ajou University) and Jee-Haeh Do (Seoul National University) for providing technical assistance. This work was supported by Glycomics Research Project Grants (No. 20100002028, GRP 2008-2012) from Korea Science and Engineering Foundation & National Research Foundation of Korea.

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Citation Excerpt :
In contrast, Ryu et al. showed that OGT activity is suppressed by S-nitrosylation and LPS activation of macrophages leads to denitrosylation of OGT and enhanced cellular protein O-GlcNAcylation. Experimental inhibition of cellular O-GlcNAcylation results in suppression of LPS-induced macrophage inflammatory response [70]. To add to this paradox, it has also been reported that LPS-induced c-Rel activation decreases under hyperglycemic condition along with its decreased O-GlcNAcylation.
O-linked β-N-acetyl glucosamine modification (O-GlcNAcylation) is a dynamic, reversible posttranslational modification of cytoplasmic and nuclear proteins. O-GlcNAcylation depends on nutrient availability and the hexosamine biosynthetic pathway (HBP), which produces the donor substrate UDP-GlcNAc. O-GlcNAcylation is mediated by a single enzyme, O-GlcNAc transferase (OGT), which adds GlcNAc and another enzyme, O-GlcNAcase (OGA), which removes O-GlcNAc from proteins. O-GlcNAcylation controls vital cellular processes including transcription, translation, the cell cycle, metabolism, and cellular stress. Aberrant O-GlcNAcylation has been implicated in various pathologies including Alzheimer’s disease, diabetes, obesity, and cancer. Growing evidences indicate that O-GlcNAcylation plays crucial roles in regulating immunity and inflammatory responses, especially under hyperglycemic conditions. This review will highlight the emerging functions of O-GlcNAcylation in mammalian immunity under physiological and various pathological conditions.

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

Abstract

Highlights

Introduction

Section snippets

Materials

Enhanced protein O-GlcNAcylation in LPS-treated macrophage cells in vitro and in vivo

Acknowledgments

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Cell

J. Biol. Chem.

J. Biol. Chem.

Methods Enzymol.

Chem. Biol.

The hexosamine signaling pathway: deciphering the ‘O -GlcNAc code’

Sci. STKE

Cycling of O-linked beta- N-acetylglucosamine on nucleocytoplasmic proteins

Nature

The superhelical TPR-repeat domain of O-linked GlcNAc transferase exhibits structural similarities to importin alpha

Nat. Struct. Mol. Biol.

Structural insights into mechanism and specificity of O-GlcNAc transferase

EMBO J.

Glycosylation of nucleocytoplasmic proteins: signal transduction and O-GlcNAc

Science

Phosphoinositide signaling links O-GlcNAc transferase to insulin resistance

Nature

Flux through the hexosamine pathway is a determinant of nuclear factor kappaB-dependent promoter activation

Diabetes

The O-linked N-acetylglucosamine modification in cellular signaling and the immune system

EMBO reports