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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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Inducible expression of mOGT in IRES bicistronic vectors leads to toxicity requiring proper targeting and catalytic activity. 1. pI-mOGT-IRES-GFP has the full length OGT gene placed after an ecdysone-inducible promoter and an internal ribosomal entry site is located between OGT and GFP. This vector is used as a backbone for the derivatives described below. 2. pI-mOGT-G-IRES-GFP contains a C-terminal deletion represented by dotted lines (ΔC-term-del). This catalytically defective mOGT replaces the full length wild-type mOGT. 3. pI-mOGT-4A-IRES-GFP contains a deletion (dotted lines) of the 15 N-terminal amino acids thought to be involved in mitochondrial targeting (MLQGHFWLREGIMIS) and has an added histidine tag (solid red stripe) at the N-terminus (ΔN-term-del + His tag). 4. pI-mOGT-4A-G-IRES-GFP is catalytically defective and contains both C-terminal and N-terminal deletions plus the histidine tag described for vectors 2 and 3, respectively. These plasmids were transfected into EcR-293 cells and the surviving colonies were selected with G418 as described. Colonies were counted to quantify the degree of toxicity for each construct. The minus sign means no colonies were observed. There is roughly an order of magnitude difference between one plus and two plus signs. Approximate colony numbers are shown. The colony count was carried out in triplicate and a summary of the three experiments is presented
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Fig1: Inducible expression of mOGT in IRES bicistronic vectors leads to toxicity requiring proper targeting and catalytic activity. 1. pI-mOGT-IRES-GFP has the full length OGT gene placed after an ecdysone-inducible promoter and an internal ribosomal entry site is located between OGT and GFP. This vector is used as a backbone for the derivatives described below. 2. pI-mOGT-G-IRES-GFP contains a C-terminal deletion represented by dotted lines (ΔC-term-del). This catalytically defective mOGT replaces the full length wild-type mOGT. 3. pI-mOGT-4A-IRES-GFP contains a deletion (dotted lines) of the 15 N-terminal amino acids thought to be involved in mitochondrial targeting (MLQGHFWLREGIMIS) and has an added histidine tag (solid red stripe) at the N-terminus (ΔN-term-del + His tag). 4. pI-mOGT-4A-G-IRES-GFP is catalytically defective and contains both C-terminal and N-terminal deletions plus the histidine tag described for vectors 2 and 3, respectively. These plasmids were transfected into EcR-293 cells and the surviving colonies were selected with G418 as described. Colonies were counted to quantify the degree of toxicity for each construct. The minus sign means no colonies were observed. There is roughly an order of magnitude difference between one plus and two plus signs. Approximate colony numbers are shown. The colony count was carried out in triplicate and a summary of the three experiments is presented

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


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

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

Inducible expression of mOGT in IRES bicistronic vectors leads to toxicity requiring proper targeting and catalytic activity. 1. pI-mOGT-IRES-GFP has the full length OGT gene placed after an ecdysone-inducible promoter and an internal ribosomal entry site is located between OGT and GFP. This vector is used as a backbone for the derivatives described below. 2. pI-mOGT-G-IRES-GFP contains a C-terminal deletion represented by dotted lines (ΔC-term-del). This catalytically defective mOGT replaces the full length wild-type mOGT. 3. pI-mOGT-4A-IRES-GFP contains a deletion (dotted lines) of the 15 N-terminal amino acids thought to be involved in mitochondrial targeting (MLQGHFWLREGIMIS) and has an added histidine tag (solid red stripe) at the N-terminus (ΔN-term-del + His tag). 4. pI-mOGT-4A-G-IRES-GFP is catalytically defective and contains both C-terminal and N-terminal deletions plus the histidine tag described for vectors 2 and 3, respectively. These plasmids were transfected into EcR-293 cells and the surviving colonies were selected with G418 as described. Colonies were counted to quantify the degree of toxicity for each construct. The minus sign means no colonies were observed. There is roughly an order of magnitude difference between one plus and two plus signs. Approximate colony numbers are shown. The colony count was carried out in triplicate and a summary of the three experiments is presented
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040817&req=5

Fig1: Inducible expression of mOGT in IRES bicistronic vectors leads to toxicity requiring proper targeting and catalytic activity. 1. pI-mOGT-IRES-GFP has the full length OGT gene placed after an ecdysone-inducible promoter and an internal ribosomal entry site is located between OGT and GFP. This vector is used as a backbone for the derivatives described below. 2. pI-mOGT-G-IRES-GFP contains a C-terminal deletion represented by dotted lines (ΔC-term-del). This catalytically defective mOGT replaces the full length wild-type mOGT. 3. pI-mOGT-4A-IRES-GFP contains a deletion (dotted lines) of the 15 N-terminal amino acids thought to be involved in mitochondrial targeting (MLQGHFWLREGIMIS) and has an added histidine tag (solid red stripe) at the N-terminus (ΔN-term-del + His tag). 4. pI-mOGT-4A-G-IRES-GFP is catalytically defective and contains both C-terminal and N-terminal deletions plus the histidine tag described for vectors 2 and 3, respectively. These plasmids were transfected into EcR-293 cells and the surviving colonies were selected with G418 as described. Colonies were counted to quantify the degree of toxicity for each construct. The minus sign means no colonies were observed. There is roughly an order of magnitude difference between one plus and two plus signs. Approximate colony numbers are shown. The colony count was carried out in triplicate and a summary of the three experiments is presented
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

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