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

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 acetyltransferases. 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.