Characterization of β-N-Acetylglucosaminidase Cleavage by Caspase-3 during Apoptosis

Chutikarn Butkinaree,Win D Cheung,Sungjin Park,Kyoungsook Park,Megan Barber,Gerald W Hart

doi:10.1074/jbc.m804116200

Chutikarn Butkinaree, Win D Cheung + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m804116200

Copy DOI

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.

Highlights

Protein function by regulating protein activity, protein-protein interaction, localization, or protein degradation [5, 6]
Incubation of recombinant O-GlcNAcase with caspase-3 resulted in cleavage of O-GlcNAcase that yielded fragments distinguishable by gel electrophoresis from that found in the untreated O-GlcNAcase in a dose-dependent manner (Fig. 1A)
We found that O-GlcNAcase was cleaved by caspase-3 into four different fragments with molecular masses of 150, 72, 70, and 60 kDa

Summary

EXPERIMENTAL PROCEDURES

Materials and Antibodies—Rabbit anti-O-GlcNAcase antibody was kindly provided by Dr Wally Whiteheart (University of Kentucky). Recombinant caspase-3 was purchased from Biomol Research Laboratories, Inc. Generation of O-GlcNAcase Antibodies—Through Sigma, we created a rabbit polyclonal antibody and a chicken polyclonal antibody specific to the N and C termini of O-GlcNAcase, respectively, using peptides purified from bacteria expressing amino acids 1– (N-terminally specific) and amino acids –916 (C-terminally specific) of the human O-GlcNAcase protein sequence. The membranes were probed with antibodies to O-GlcNAc (1:5,000), O-GlcNAcase (anti-full-length O-GlcNAcase; 1:5000; anti-N-terminal O-GlcNAcase; 1:1,000; anti-C-terminal O-GlcNAcase; 1:1,000), PARP (1:5,000), HA (1:5,000), c-Myc (1:5,000), and caspase-3 (1:2,500) overnight at 4 °C. The day, the mixtures were incubated with IgY- Caspase-3 in Vitro Cleavage Assay—Caspase-3 cleavage agarose beads (Gallus Immunotech, Inc.) for 2 h at 4 °C. the assays were performed as previously described with some modbeads were washed with cell extraction buffer Note that one unit of caspase-3 was equal to 1 pmol/min at 30 °C using Ac-DEVD-pNA as substrate

RESULTS

NAcase lies in between the folded

We found that in vitro cleavage of

DISCUSSION