The New Chemical Reporter 6‑Alkynyl-6-deoxy-GlcNAc Reveals O‑GlcNAc Modification of the Apoptotic Caspases That Can Block the Cleavage/Activation of Caspase‑8

• Published in: Journal of the American Chemical Society Vol. 139; no. 23; pp. 7872 - 7885
• Main Authors: Chuh, Kelly N, Batt, Anna R, Zaro, Balyn W, Darabedian, Narek, Marotta, Nicholas P, Brennan, Caroline K, Amirhekmat, Arya, Pratt, Matthew R
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 14-06-2017
• Subjects: Acetylglucosamine - chemistry; Acetylglucosamine - metabolism; Animals; Apoptosis; Caspases - chemistry; Caspases - metabolism; Glycosylation; Humans; Kinetics; MCF-7 Cells; Mice; NIH 3T3 Cells
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/jacs.7b02213

O-GlcNAc modification (O-GlcNAcylation) is required for survival in mammalian cells. Genetic and biochemical experiments have found that increased modification inhibits apoptosis in tissues and cell culture and that lowering O-GlcNAcylation induces cell death. However, the molecular mechanisms by which O-GlcNAcylation might inhibit apoptosis are still being elucidated. Here, we first synthesize a new metabolic chemical reporter, 6-Alkynyl-6-deoxy-GlcNAc (6AlkGlcNAc), for the identification of O-GlcNAc-modified proteins. Subsequent characterization of 6AlkGlcNAc shows that this probe is selectively incorporated into O-GlcNAcylated proteins over cell-surface glycoproteins. Using this probe, we discover that the apoptotic caspases are O-GlcNAcylated, which we confirmed using other techniques, raising the possibility that the modification affects their biochemistry. We then demonstrate that changes in the global levels of O-GlcNAcylation result in a converse change in the kinetics of caspase-8 activation during apoptosis. Finally, we show that caspase-8 is modified at residues that can block its cleavage/activation. Our results provide the first evidence that the caspases may be directly affected by O-GlcNAcylation as a potential antiapoptotic mechanism.