OGT functions as a catalytic chaperone under heat stress response: a unique defense role of OGT in hyperthermia

doi:10.1016/j.bbrc.2004.08.023

Biochemical and Biophysical Research Communications

Volume 322, Issue 3, 24 September 2004, Pages 1045-1051

https://doi.org/10.1016/j.bbrc.2004.08.023 Get rights and content

Abstract

Protein O-GlcNAcylation is proceeded by O-linked GlcNAc transferase (OGT) in nucleocytoplasm and is involved in many biological processes although its physiological role is not clearly defined. To identify the functional significance of O-GlcNAcylation, we investigated heat stress effects on protein O-GlcNAcylation. Here, we found that protein O-GlcNAcylation was significantly increased in vivo during acute heat stress in mammalian cells and simultaneously, the enhanced protein O-GlcNAcylation was closely associated with cell survival in hyperthermia. Our results demonstrate that hyperthermal cytotoxicity may considerably be facilitated under the condition of insufficient level of protein O-GlcNAcylation inside cells. Furthermore, OGT reaction might be crucial for triggering thermotolerance to recover hyperthermal sensitivity without particular induction of heat shock proteins (hsps). Thus, we propose that OGT can respond rapidly to heat stress through the enhancement of nucleocytoplasmic protein O-GlcNAcylation for a rescue from the early phase of hyperthermal cytotoxicity.

Section snippets

Experimental procedures

Cell cultures. Cell culture reagents were purchased from Gibco-BRL. CHO-K1 (Chinese hamster ovary) cells and Hep3B (human hepatocarcinoma) cells were obtained from ATCC. CHO-K1 cells were cultured in α-MEM containing 10% fetal bovine serum (FBS) supplemented with penicillin and streptomycin antibiotics. Hep3B cells were maintained in DMEM containing 10% FBS supplemented with antibiotics. Cells were grown as monolayers at 37 °C in a humidified incubator with 5% CO₂ in air to maintain medium pH at

Heat stress treatment and analysis of protein O-GlcNAcylation

To examine the effect of heat stress on protein O-GlcNAcylation in mammalian cells, we incubated CHO cells and Hep3B cells, at 45 °C, for various time periods ranging from 0 to 75 min and the level of protein O-GlcNAcylation was analyzed by immunoblotting. Notably, protein O-GlcNAcylation was significantly and rapidly enhanced during heat stress treatment in both cells (Figs. 1A and B). This finding indicates that the increment of protein O-GlcNAcylation appears to be closely linked to heat

Discussion

OGT contains a TPR domain in N-terminal half and a CAT domain in C-terminal half [30], [36]. In particular, TPR domain consisted of 11.5 TPRs and is known to be involved in substrate recognition by protein–protein interaction [37], [38], although molecular basis of the substrate recognition is poorly understood. Based on a protein recognition function of OGT, we considered the possibility that OGT might have a defensive role against heat stress largely through its protein O-GlcNAcylation. In

Acknowledgments

We thank Dr. K.-H. you (Chungnam National Univeristy) and Dr. S.-S. Lee (Pai Chai University) for technical assistance. This work was supported by Ajou Research Grants” (ARG) from Ajou University (P.S. Hwang, ARG 20033390 and ARG 20033270) and Glycomics Research Project (MOST, 2004).