Elevated O-GlcNAc-dependent signaling through inducible mOGT expression selectively triggers apoptosis.
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.
Affiliation: Gyeong-Gi Bio Center, Suwon, Korea.
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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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