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: Previously, we showed that transient overexpression of the mitochondrial form of OGT (mOGT) increased cellular OGT activity, but was cytotoxic (Hanover et al. 2003; Lubas et al. 1997). Overexpression of the ncOGT isoform did not show obvious cytotoxicity. To understand the basis for the selective cytotoxicity, we have developed a conditional expression system of OGT in mammalian cell lines. We used an ecdysone-inducible gene expression system (outlined in Fig. 1). To identify the subtle difference of OGT expression in the cells, we first added the green fluorescence protein (GFP) as a second marker and an internal ribosomal entry site (IRES). When this construct was transfected into cells, in the presence of the ecdysone analog ponasterone A, the ecdysone-dependent inducible promoter directed the production of a bicistronic transcript encoding two proteins: mOGT and GFP, which is translated independently using the IRES. We observed colonies from stably transfected cells under selective pressure of G418 in the absence of ponasterone A. We could not generate a stable cell line from the dishes transfected with pI-mOGT-IRES-GFP. However, we got stable cell lines stably expressing the inactive pI-mOGT-G-IRES-GFP, containing C-terminal deletion. Interestingly, we observed an order of magnitude higher number of colonies derived from either pI-4A-mOGT-IRES-GFP (containing a N-terminal deletion) or pI-4A-mOGT-G-IRES-GFP (containing both N-and C-terminal deletions) (Fig. 1) in comparison with the dishes transfected with pI-mOGT-G-IRES-GFP. With bicistronic constructs, we saw a minimal level of transient or stable expression of either mOGT or GFP in the absence of ponasterone A. These experiments suggested that upon removal of the 15 amino acid N-terminus of mOGT (pI-4A-mOGT-IRES-GFP), or by inactivating the catalytic domain of mOGT by truncation (pI-mOGT-G-IRES-GFP), much of the toxicity of mOGT was eliminated.Fig. 1