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

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.