Characterization of beta-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 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.
Butkinaree
Chutikarn
C
Departments of Biological Chemistry and Neuroscience, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205-2185, USA.
Cheung
Win D
WD
Park
Sungjin
S
Park
Kyoungsook
K
Barber
Megan
M
Hart
Gerald W
GW
D000117:Acetylglucosamine / Q000235:genetics / Q000378:metabolism; D000118:Acetylglucosaminidase / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D017209:Apoptosis / Q000502:physiology*; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D019169:Jurkat Cells; D017354:Point Mutation; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D017434:Protein Structure, Tertiary / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity / Q000502:physiology; D050880:p300-CBP Transcription Factors / Q000235:genetics / Q000378:metabolism; D000099224:p300-CBP-Associated Factor
D000117:Acetylglucosamine; D000118:Acetylglucosaminidase; D053148:Caspase 3; D050880:p300-CBP Transcription Factors; D000099224:p300-CBP-Associated Factor; C505407:CASP3 protein, human