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Elevated O-GlcNAc-dependent signaling through inducible mOGT expression selectively triggers apoptosis.

Shin SH, Love DC, Hanover JA - Amino Acids (2010)

Bottom Line: One differentially spliced isoform of OGT is normally targeted to mitochondria (mOGT) but is quite cytotoxic when expressed in cells compared with the ncOGT isoform.Apoptosis was dependent upon the unique N-terminus of mOGT, and its catalytic activity.Induction of mOGT expression triggered programmed cell death in every cell type tested including INS-1, an insulin-secreting cell line.

View Article: PubMed Central - PubMed

Affiliation: Gyeong-Gi Bio Center, Suwon, Korea.

ABSTRACT
O-linked N-acetylglucosamine transferase (OGT) catalyzes O-GlcNAc addition to numerous cellular proteins including transcription and nuclear pore complexes and plays a key role in cellular signaling. One differentially spliced isoform of OGT is normally targeted to mitochondria (mOGT) but is quite cytotoxic when expressed in cells compared with the ncOGT isoform. To understand the basis of this selective cytotoxicity, we constructed a fully functional ecdysone-inducible GFP-OGT. Elevated GFP-OGT expression induced a dramatic increase in intracellular O-GlcNAcylated proteins. Furthermore, enhanced OGT expression efficiently triggered programmed cell death. Apoptosis was dependent upon the unique N-terminus of mOGT, and its catalytic activity. Induction of mOGT expression triggered programmed cell death in every cell type tested including INS-1, an insulin-secreting cell line. These studies suggest that deregulated activity of the mitochondrially targeted mOGT may play a role in triggering the programmed cell death observed with diseases such as diabetes mellitus and neurodegeneration.

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O-GlcNAcylation is consistent with GFP-fused OGT expression, but not with mutant, catalytically defective, OGT. a Western blot analysis reveals that induction of GFP-fused OGT expression with increasing concentrations of Ponasterone A (PoA) (0.5, 3, 15 µM, respectively) causes an increase in the levels of total O-GlcNAc modification. The level of O-GlcNAc modified proteins prior to induction is shown in the last lane (−). The anti-O-GlcNAc antibody, RL2, was used in the Western Blot analysis. b Immunofluorescent detection of GFP-fused OGT expression and its catalytic activity using RL2 staining. RL2 specifically recognizes O-GlcNAcylated proteins. pGFP–OGT-G is the c-terminal deleted, catalytically defective OGT under CMV-driven promoter
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Fig3: O-GlcNAcylation is consistent with GFP-fused OGT expression, but not with mutant, catalytically defective, OGT. a Western blot analysis reveals that induction of GFP-fused OGT expression with increasing concentrations of Ponasterone A (PoA) (0.5, 3, 15 µM, respectively) causes an increase in the levels of total O-GlcNAc modification. The level of O-GlcNAc modified proteins prior to induction is shown in the last lane (−). The anti-O-GlcNAc antibody, RL2, was used in the Western Blot analysis. b Immunofluorescent detection of GFP-fused OGT expression and its catalytic activity using RL2 staining. RL2 specifically recognizes O-GlcNAcylated proteins. pGFP–OGT-G is the c-terminal deleted, catalytically defective OGT under CMV-driven promoter

Mentions: To confirm that the GFP–OGT fusion protein was catalytically active, the levels of O-GlcNAc were examined using anti-O-GlcNAc antibodies and immunoblotting and immunofluorescence microscopy (Fig. 3). Increasing concentrations of Ponasterone A lead to increased O-GlcNAc levels on several cellular proteins (Fig. 3a). By immunofluorescence, we observed a substantial increase in O-GlcNAc in those cells induced to express the GFP–OGT fusion protein; no elevation was observed in those cells not expressing the fusion protein (Fig. 3b, pIND/GFP–OGT). Similar results were observed when a constitutive CMV promoter (pGFP–OGT) was used (Fig. 3b, pGFP–OGT). When we expressed a GFP–OGT fusion with a truncation in the catalytic domain, which renders it inactive (Lubas and Hanover 2000), O-GlcNAc levels were not elevated (Fig. 3b, pGFP–OGT-G). These data suggest that GFP–OGT is catalytically active when expressed in cells; this activity requires an intact OGT catalytic domain.Fig. 3


Elevated O-GlcNAc-dependent signaling through inducible mOGT expression selectively triggers apoptosis.

Shin SH, Love DC, Hanover JA - Amino Acids (2010)

O-GlcNAcylation is consistent with GFP-fused OGT expression, but not with mutant, catalytically defective, OGT. a Western blot analysis reveals that induction of GFP-fused OGT expression with increasing concentrations of Ponasterone A (PoA) (0.5, 3, 15 µM, respectively) causes an increase in the levels of total O-GlcNAc modification. The level of O-GlcNAc modified proteins prior to induction is shown in the last lane (−). The anti-O-GlcNAc antibody, RL2, was used in the Western Blot analysis. b Immunofluorescent detection of GFP-fused OGT expression and its catalytic activity using RL2 staining. RL2 specifically recognizes O-GlcNAcylated proteins. pGFP–OGT-G is the c-terminal deleted, catalytically defective OGT under CMV-driven promoter
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3040817&req=5

Fig3: O-GlcNAcylation is consistent with GFP-fused OGT expression, but not with mutant, catalytically defective, OGT. a Western blot analysis reveals that induction of GFP-fused OGT expression with increasing concentrations of Ponasterone A (PoA) (0.5, 3, 15 µM, respectively) causes an increase in the levels of total O-GlcNAc modification. The level of O-GlcNAc modified proteins prior to induction is shown in the last lane (−). The anti-O-GlcNAc antibody, RL2, was used in the Western Blot analysis. b Immunofluorescent detection of GFP-fused OGT expression and its catalytic activity using RL2 staining. RL2 specifically recognizes O-GlcNAcylated proteins. pGFP–OGT-G is the c-terminal deleted, catalytically defective OGT under CMV-driven promoter
Mentions: To confirm that the GFP–OGT fusion protein was catalytically active, the levels of O-GlcNAc were examined using anti-O-GlcNAc antibodies and immunoblotting and immunofluorescence microscopy (Fig. 3). Increasing concentrations of Ponasterone A lead to increased O-GlcNAc levels on several cellular proteins (Fig. 3a). By immunofluorescence, we observed a substantial increase in O-GlcNAc in those cells induced to express the GFP–OGT fusion protein; no elevation was observed in those cells not expressing the fusion protein (Fig. 3b, pIND/GFP–OGT). Similar results were observed when a constitutive CMV promoter (pGFP–OGT) was used (Fig. 3b, pGFP–OGT). When we expressed a GFP–OGT fusion with a truncation in the catalytic domain, which renders it inactive (Lubas and Hanover 2000), O-GlcNAc levels were not elevated (Fig. 3b, pGFP–OGT-G). These data suggest that GFP–OGT is catalytically active when expressed in cells; this activity requires an intact OGT catalytic domain.Fig. 3

Bottom Line: One differentially spliced isoform of OGT is normally targeted to mitochondria (mOGT) but is quite cytotoxic when expressed in cells compared with the ncOGT isoform.Apoptosis was dependent upon the unique N-terminus of mOGT, and its catalytic activity.Induction of mOGT expression triggered programmed cell death in every cell type tested including INS-1, an insulin-secreting cell line.

View Article: PubMed Central - PubMed

Affiliation: Gyeong-Gi Bio Center, Suwon, Korea.

ABSTRACT
O-linked N-acetylglucosamine transferase (OGT) catalyzes O-GlcNAc addition to numerous cellular proteins including transcription and nuclear pore complexes and plays a key role in cellular signaling. One differentially spliced isoform of OGT is normally targeted to mitochondria (mOGT) but is quite cytotoxic when expressed in cells compared with the ncOGT isoform. To understand the basis of this selective cytotoxicity, we constructed a fully functional ecdysone-inducible GFP-OGT. Elevated GFP-OGT expression induced a dramatic increase in intracellular O-GlcNAcylated proteins. Furthermore, enhanced OGT expression efficiently triggered programmed cell death. Apoptosis was dependent upon the unique N-terminus of mOGT, and its catalytic activity. Induction of mOGT expression triggered programmed cell death in every cell type tested including INS-1, an insulin-secreting cell line. These studies suggest that deregulated activity of the mitochondrially targeted mOGT may play a role in triggering the programmed cell death observed with diseases such as diabetes mellitus and neurodegeneration.

Show MeSH
Related in: MedlinePlus