Characterization of beta-N-acetylglucosaminidase cleavage by caspase-3 during apoptosis

Abstract

Beta-O-linked N-acetylglucosamine is a dynamic post-translational modification involved in protein regulation in a manner similar to phosphorylation. Removal of N-acetylglucosamine is regulated by beta-N-acetylglucosaminidase (O-GlcNAcase), which was previously shown to be a substrate of caspase-3 in vitro. Here we show that O-GlcNAcase is cleaved by caspase-3 into two fragments during apoptosis, an N-terminal fragment containing the O-GlcNAcase active site and a C-terminal fragment containing a region with homology to GCN5 histone acetyl-transferases. The caspase-3 cleavage site of O-GlcNAcase, mapped by Edman sequencing, is a noncanonical recognition site that occurs after Asp-413 of the SVVD sequence in human O-GlcNAcase. A point mutation, D413A, abrogates cleavage by caspase-3 both in vitro and in vivo. Finally, we show that O-GlcNAcase activity is not affected by caspase-3 cleavage because the N- and C-terminal O-GlcNAcase fragments remain associated after the cleavage. Furthermore, when co-expressed simultaneously in the same cell, the N-terminal and C-terminal caspase fragments associate to reconstitute O-GlcNAcase enzymatic activity. These studies support the identification of O-GlcNAcase as a caspase-3 substrate with a novel caspase-3 cleavage site and provide insight about O-GlcNAcase regulation during apoptosis.

FIGURE 1.

Cleavage of recombinant O-GlcNAcase by caspase-3 in vitro. A, 5 μg of human recombinant O-GlcNAcase was incubated with indicated amounts of recombinant caspase-3 in caspase-3 assay buffer at 37 °C for 2.5 h. The reaction was stopped by adding Laemmli buffer for Coomassie G-250 staining and Western analysis or 100 μm of caspase-3 inhibitor Ac-DEVD-CHO for O-GlcNAcase activity assay. Cleavage mixtures from in vitro caspase-3 cleavage assay were subjected to 7.5% SDS-PAGE followed by Coomassie G-250 staining. B, recombinant O-GlcNAcase was incubated with 200 units of caspase-3 in the absence or presence of various concentrations (as indicated) of peptide aldehyde inhibitor Ac-DEVD-CHO and subjected to 7.5% SDS-PAGE followed by immunoblotting (IB) against O-GlcNAcase antibody. C, cleavage mixtures from caspase-3 cleavage assay using various amounts of caspase-3 as indicated was analyzed for O-GlcNAcase activity in triplicate.

FIGURE 2.

O-GlcNAcase is cleaved in cells undergoing apoptosis. A, Jurkat cells were treated with 100 ng/ml of either anti-mouse IgM (control) or anti-Fas CH11 mAb for different time periods. The cell lysates were subjected to 7.5% SDS-PAGE followed by Western blot analysis for O-GlcNAcase. Immunoblotting (IB) with PARP and actin antibodies were used as a control for apoptosis and loading respectively. B, O-GlcNAc levels in Jurkat cells undergoing Fas-mediated apoptosis. C, Jurkat cells were treated with 150 μm H₂O₂ or PBS (as control) for 8 h. The cell lysates were subjected to Western blot analysis with C-terminally specific O-GlcNAcase, PARP, and actin antibodies.

FIGURE 3.

O-GlcNAcase is cleaved by caspase-3 during Fas-mediated apoptosis, and caspase-3 cleavage during apoptosis does not affect O-GlcNAcase enzymatic activity. A, Jurkat cells were treated with 100 ng/ml of either anti-mouse IgM (control) or anti-Fas CH11 mAb in the presence of dimethyl sulfoxide (DMSO, control) or 50 μm Z-VAD-fmk or 100 μm Z-DEVD-fmk for 6 h, harvested, and subjected to SDS-PAGE, and Western blot analysis for O-GlcNAcase cleavage and apoptosis using O-GlcNAcase, PARP, and actin antibodies. B, 20 μg of lysate from each sample were analyzed for O-GlcNAcase activity in triplicate.

FIGURE 4.

Recombinant O-GlcNAcase is cleaved by caspase-3 after Asp-413 of the tetrapeptide sequence SVVD in human O-GlcNAcase amino acid sequence. 120 μg of recombinant O-GlcNAcase was incubated with 2500 units of caspase-3 at 37 °C for 2.5 h. The cleavage mixtures were subjected to 7.5% SDS-PAGE and transferred onto a polyvinylidene difluoride membrane, which was later stained with Coomassie R-250. Four bands that appeared in the cleavage mixtures but not in the untreated O-GlcNAcase were cut out and sequenced in an automated amino acid sequencer (see Fig. 1A for the four fragments used in this analysis). A, repetitive yield from N-terminal sequencing indicating the first seven amino acids in the N-terminal sequence of fragment 2. B, schematic view of caspase-3 cleavage of recombinant O-GlcNAcase, where the splice variant is also shown (25, 54). Trx·tag, thioredoxin tag. C, diagram showing the predicted folded and unfolded regions of human O-GlcNAcase using the FoldIndex program (49). D, wild-type O-GlcNAcase and D413A mutant were subjected to an in vitro caspase-3 cleavage assay. Cleavage products were analyzed by immunoblotting (IB) with O-GlcNAcase antibody.

FIGURE 5.

The point mutation D413A abrogates cleavage of O-GlcNAcase during apoptosis in vivo. HeLa cells transfected with empty vector, wild-type O-GlcNAcase, or point-mutated D413A O-GlcNAcase were treated with 1 μg/ml of either anti-mouse IgM or anti-Fas mAb for 4 h. The cell lysates were subjected to SDS-PAGE and Western blot analysis for O-GlcNAcase. IB, immunoblotting.

FIGURE 6.

Both the N and C termini of O-GlcNAcase are required for O-GlcNAcase activity. HeLa cells were transfected with DNA from empty pRK5 vector, full-length (FL) O-GlcNAcase, N-terminal O-GlcNAcase (N, amino acids 1–413), C-terminal O-GlcNAcase (C, amino acids 414–916), or both N- and C-terminal O-GlcNAcase plasmids (N+C). pRK5 vector, full-length O-GlcNAcase, and the N-terminal O-GlcNAcase plasmids contained c-Myc tag, whereas the C-terminal O-GlcNAcase plasmids contained the HA tag. The cells were harvested after 1 day of transfection, extracted, and analyzed for O-GlcNAcase expression and O-GlcNAcase activity. A, O-GlcNAcase activity from transfected HeLa cells. Plasmids and amount of DNA used (for co-transfection of O-GlcNAcase N and C termini, in μg) are indicated on the x axis. B and C, cell extracts were subjected to either native PAGE (B) or SDS-PAGE (C) and Western blot analysis for O-GlcNAcase using anti-HA and anti-c-Myc antibodies (left and right panels, respectively). Actin detection was used as loading controls. The plasmids and amounts of DNA used were as indicated. IB, immunoblotting.

FIGURE 7.

The N and C termini of O-GlcNAcase remain associated after caspase-3 cleavage. After a 1-day transfection, HeLa cells were treated with 1 μg/ml of either anti-mouse IgM (control) or anti-Fas CH11 mAb for 4 h. The cell extracts were subjected to immunoprecipitation using antibodies specific to the C terminus of O-GlcNAcase. Immunoprecipitated products were subjected to Western blot analysis using antibodies specific to the C-terminal O-GlcNAcase (upper panel) and the N-terminal O-GlcNAcase (lower panel). 1°, control immunoprecipitation of anti-C-terminal O-GlcNAcase antibody in blank extraction buffer. IgY, control immunoprecipitation using anti-chicken IgY antibody instead of anti-C-terminal O-GlcNAcase antibody. FL, full length. N+C, N and C termini. IB, immunoblotting.