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J. Biol. Chem., Vol. 283, Issue 35, 23557-23566, August 29, 2008

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

Chutikarn Butkinaree, Win D. Cheung, Sungjin Park, Kyoungsook Park, Megan Barber, and Gerald W. Hart¹

From the Departments of Biological Chemistry and Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185

β-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 β-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.

Received for publication, May 29, 2008 , and in revised form, June 26, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grants HD13563 and CA42486 (to G. W. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S6.

¹ Supported in part by a share of royalties received by the university on sales of the CTD 110.6 antibody. To whom correspondence should be addressed: Dept. of Biological Chemistry, Johns Hopkins University School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205-2185. Tel.: 410-614-5993; Fax: 410-614-8804; E-mail: gwhart{at}jhmi.edu.

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