O-GlcNAcase Expression is Sensitive to Changes in O-GlcNAc Homeostasis

Abstract

O-linked N-acetylglucosamine (O-GlcNAc) is a post-translational modification involving an attachment of a single β-N-acetylglucosamine moiety to serine or threonine residues in nuclear and cytoplasmic proteins. Cellular O-GlcNAc levels are regulated by two enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which add and remove the modification, respectively. The levels of O-GlcNAc can rapidly change in response to fluctuations in the extracellular environment; however, O-GlcNAcylation returns to a baseline level quickly after stimulus removal. This process termed O-GlcNAc homeostasis appears to be critical to the regulation of many cellular functions including cell cycle progress, stress response, and gene transcription. Disruptions in O-GlcNAc homeostasis are proposed to lead to the development of diseases, such as cancer, diabetes, and Alzheimer's disease. O-GlcNAc homeostasis is correlated with the expression of OGT and OGA. We reason that alterations in O-GlcNAc levels affect OGA and OGT transcription. We treated several human cell lines with Thiamet-G (TMG, an OGA inhibitor) to increase overall O-GlcNAc levels resulting in decreased OGT protein expression and increased OGA protein expression. OGT transcript levels slightly declined with TMG treatment, but OGA transcript levels were significantly increased. Pretreating cells with protein translation inhibitor cycloheximide did not stabilize OGT or OGA protein expression in the presence of TMG; nor did TMG stabilize OGT and OGA mRNA levels when cells were treated with RNA transcription inhibitor actinomycin D. Finally, we performed RNA Polymerase II chromatin immunoprecipitation at the OGA promoter and found that RNA Pol II occupancy at the transcription start site was lower after prolonged TMG treatment. Together, these data suggest that OGA transcription was sensitive to changes in O-GlcNAc homeostasis and was potentially regulated by O-GlcNAc.

Keywords: O-GlcNAc; O-GlcNAc transferase; O-GlcNAcase; post-translational modification; transcription.

Figure 1

OGA protein level was increased after TMG treatment. (A) SH-SY5Y neuroblastoma cells. (B) HeLa cervical cells. (C) K562 leukemia cells were treated with 10 μM TMG for indicated time. (D) SH-SY5Y cells were infected with GFP, OGT, and OGA adenovirus at 75 MOI for 24 h. Cells were lysed, overall O-GlcNAc level, OGA and OGT protein level were analyzed by western blot with actin as a loading control. Average fold change for OGT and OGA is listed on the blots.

Figure 2

TMG does not stabilize OGA protein. (A) HeLa cells and (B) K562 cells were treated with TMG, CHX (protein translation inhibitor), and CHX + TMG. Cells were lysed, overall O-GlcNAc level, OGA and OGT protein level were analyzed by western blot, with actin as loading control.

Figure 3

OGA mRNA level was increased after TMG treatment. After TMG treatment, relative OGA mRNA level in (A) SH-SY5Y, (B) HeLa, and (C) K562 cells, as well as OGT mRNA level in (D) SH-SY5Y, (E) HeLa, and (F) K562 cells was analyzed by qPCR. (G) OGA mRNA level and (H) OGT mRNA level in SH-SY5Y cells infected with GFP, OGT, and OGA adenovirus at 75 MOI for 24 h, respectively, were also analyzed by qPCR. Hypoxanthine- guanine phosphoribosyltransferase (HPRT) was served as internal control. *P < 0.05. **P < 0.01, compared with control (TMG 0 h or GFP), n = 3, Student’s t-test.

Figure 4

TMG does not stabilize OGA mRNA. HeLa cells (A,B) and K562 cells (C,D) were treated with TMG, AMD (RNA transcription inhibitor), and AMD + TMG, respectively. (A) OGA (A,C) and OGT (B,D) mRNA level were analyzed by qPCR, with HPRT as internal control. *P < 0.05. **P < 0.01, compared with control, n = 3, Student’s t-test.

Figure 5

RNA Pol II occupancy at OGA TSS was decreased after 48 h TMG treatment in K562 cells. (A) RNA Pol II ChIP assay was performed on control and 48 h TMG treated cells. ChIP DNA was analyzed by qPCR using a set of primer targeting 1000 bp upstream of OGA TSS (−1000), OGA TSS (0), and +700 bp downstream of OGA TSS (+2700). *P < 0.05, n = 3, Student’s t-test. (B) Normal mouse IgG ChIP served as a negative control.