Optimization of chemoenzymatic mass-tagging by strain-promoted cycloaddition (SPAAC) for the determination of O-GlcNAc stoichiometry by Western blotting

The dynamic modification of intracellular proteins by O-linked β-N-acetylglucosamine (O-GlcNAcylation) plays critical roles in many cellular processes. Although various methods have been developed for O-GlcNAc detection, there are few techniques for monitoring glycosylation stoichiometry and state (i.e., mono-, di-, etc., O-GlcNAcylated). Measuring the levels of O-GlcNAcylation on a given substrate protein is important for understanding the biology of this critical modification and for prioritizing substrates for functional studies. One powerful solution to this limitation involves the chemoenzymatic installation of polyethylene glycol polymers of defined molecular mass onto O-GlcNAcylated proteins. These “mass tags” produce shifts in protein migration during sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) that can be detected by Western blotting. Broad adoption of this method by the scientific community has been limited, however, by a lack of commercially available reagents and well-defined protein standards. Here, we develop a “click chemistry” approach to this method using entirely commercial reagents and confirm the accuracy of the approach using a semisynthetic O-GlcNAcylated protein. Our studies establish a new, expedited experimental workflow and standardized methods that can be readily utilized by non-experts to quantify the O-GlcNAc stoichiometry and state on endogenous proteins in any cell or tissue lysate

Optimization of chemoenzymatic mass-tagging by strain-promoted cycloaddition (SPAAC) for the determination of O-GlcNAc stoichiometry by Western blotting

The dynamic modification of intracellular proteins by O-linked β-N-acetylglucosamine (O-GlcNAcylation) plays critical roles in many cellular processes. Although various methods have been developed for O-GlcNAc detection, there are few techniques for monitoring glycosylation stoichiometry and state (i.e., mono-, di-, etc., O-GlcNAcylated). Measuring the levels of O-GlcNAcylation on a given substrate protein is important for understanding the biology of this critical modification and for prioritizing substrates for functional studies. One powerful solution to this limitation involves the chemoenzymatic installation of polyethylene glycol polymers of defined molecular mass onto O-GlcNAcylated proteins. These “mass tags” produce shifts in protein migration during sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) that can be detected by Western blotting. Broad adoption of this method by the scientific community has been limited, however, by a lack of commercially available reagents and well-defined protein standards. Here, we develop a “click chemistry” approach to this method using entirely commercial reagents and confirm the accuracy of the approach using a semisynthetic O-GlcNAcylated protein. Our studies establish a new, expedited experimental workflow and standardized methods that can be readily utilized by non-experts to quantify the O-GlcNAc stoichiometry and state on endogenous proteins in any cell or tissue lysate

Le syndrome de Capgras (Réflexion clinique et épistémologique)

BESANCON-BU Médecine pharmacie (250562102) / SudocSudocFranceF

The Metabolic Chemical Reporter 6‑Azido-6-deoxy-glucose Further Reveals the Substrate Promiscuity of <i>O</i>‑GlcNAc Transferase and Catalyzes the Discovery of Intracellular Protein Modification by <i>O</i>‑Glucose

Metabolic chemical reporters of glycosylation in combination with bioorthogonal reactions have been known for two decades and have been used by many different research laboratories for the identification and visualization of glycoconjugates. More recently, however, they have begun to see utility for the investigation of cellular metabolism and the tolerance of biosynthetic enzymes and glycosyltransferases to different sugars. Here, we take this concept one step further by using the metabolic chemical reporter 6-azido-6-deoxy-glucose (6AzGlc). We show that treatment of mammalian cells with the per-<i>O</i>-acetylated version of 6AzGlc results in robust labeling of a variety of proteins. Notably, the pattern of this labeling was consistent with <i>O</i>-GlcNAc modifications, suggesting that the enzyme <i>O</i>-GlcNAc transferase is quite promiscuous for its donor sugar substrates. To confirm this possibility, we show that 6AzGlc-treatment results in the labeling of known <i>O</i>-GlcNAcylated proteins, that the UDP-6AzGlc donor sugar is indeed produced in living cells, and that recombinant OGT will accept UDP-6AzGlc as a substrate in vitro. Finally, we use proteomics to first identify several bona fide 6AzGlc-modifications in mammalian cells and then an endogenous <i>O</i>-glucose modification on host cell factor. These results support the conclusion that OGT can endogenously modify proteins with both <i>N</i>-acetyl-glucosamine and glucose, raising the possibility that intracellular <i>O</i>-glucose modification may be a widespread modification under certain conditions or in particular tissues

The Metabolic Chemical Reporter 6‑Azido-6-deoxy-glucose Further Reveals the Substrate Promiscuity of <i>O</i>‑GlcNAc Transferase and Catalyzes the Discovery of Intracellular Protein Modification by <i>O</i>‑Glucose

Metabolic chemical reporters of glycosylation in combination with bioorthogonal reactions have been known for two decades and have been used by many different research laboratories for the identification and visualization of glycoconjugates. More recently, however, they have begun to see utility for the investigation of cellular metabolism and the tolerance of biosynthetic enzymes and glycosyltransferases to different sugars. Here, we take this concept one step further by using the metabolic chemical reporter 6-azido-6-deoxy-glucose (6AzGlc). We show that treatment of mammalian cells with the per-<i>O</i>-acetylated version of 6AzGlc results in robust labeling of a variety of proteins. Notably, the pattern of this labeling was consistent with <i>O</i>-GlcNAc modifications, suggesting that the enzyme <i>O</i>-GlcNAc transferase is quite promiscuous for its donor sugar substrates. To confirm this possibility, we show that 6AzGlc-treatment results in the labeling of known <i>O</i>-GlcNAcylated proteins, that the UDP-6AzGlc donor sugar is indeed produced in living cells, and that recombinant OGT will accept UDP-6AzGlc as a substrate in vitro. Finally, we use proteomics to first identify several bona fide 6AzGlc-modifications in mammalian cells and then an endogenous <i>O</i>-glucose modification on host cell factor. These results support the conclusion that OGT can endogenously modify proteins with both <i>N</i>-acetyl-glucosamine and glucose, raising the possibility that intracellular <i>O</i>-glucose modification may be a widespread modification under certain conditions or in particular tissues

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