Abstract
Monosaccharide analogs bearing bioorthogonal functionalities, or metabolic chemical reporters (MCRs) of glycosylation, have been used for approximately two decades for the visualization and identification of different glycoproteins. More recently, proteomics analyses have shown that per-O-acetylated MCRs can directly and chemically react with cysteine residues in lysates and potentially cells, drawing into question the physiological relevance of the labeling. Here, we report robust metabolism-dependent labeling by Ac42AzMan but not the structurally similar Ac44AzGal. However, the levels of background chemical-labeling of cell lysates by both reporters are low and identical. We then characterized Ac42AzMan labeling and found that the vast majority of the labeling occurs on intracellular proteins but that this MCR is not converted to previously characterized reporters of intracellular O-GlcNAc modification. Additionally, we used isotope targeted glycoproteomics (IsoTaG) proteomics to show that essentially all of the Ac42AzMan labeling is on cysteine residues. Given the implications this result has for the identification of intracellular O-GlcNAc modifications using MCRs, we then performed a meta-analysis of the potential O-GlcNAcylated proteins identified by different techniques. We found that many of the proteins identified by MCRs have also been found by other methods. Finally, we randomly selected four proteins that had only been identified as O-GlcNAcylated by MCRs and showed that half of them were indeed modified. Together, these data indicate that the selective metabolism of certain MCRs is responsible for S-glycosylation of proteins in the cytosol and nucleus. However, these results also show that MCRs are still good tools for unbiased identification of glycosylated proteins, as long as complementary methods are employed for confirmation.
Highlights
Cellular biosynthetic pathways have been exploited for over two decades to incorporate chemical functionality into proteins and posttranslational modifications (Chuh et al, 2016; Gilormini et al, 2018; Parker and Pratt, 2020)
We demonstrated that 6-azido-6-deoxy-N-acetylglucosamine (6AzGlcNAc) can bypass the traditional GlcNAc-salvage pathway to generate uridine diphosphate sugar (UDP-6AzGlcNAc) (Chuh et al, 2014), resulting in labeling of O-GlcNAcylated proteins and suggesting that cellular metabolism is more accommodating to metabolic chemical reporters (MCRs) than previously appreciated
In order to further explore the potential of structural diverse monosaccharide analogs as potential MCRs, we purchased Ac42AzMan and synthesized Ac44AzGal in a two-step, one-pot synthesis from commercially available 1,2,3,6-O-acetyl-glucose (Supporting Information)
Summary
Cellular biosynthetic pathways have been exploited for over two decades to incorporate chemical functionality into proteins and posttranslational modifications (Chuh et al, 2016; Gilormini et al, 2018; Parker and Pratt, 2020). We demonstrated that 6-azido-6-deoxy-N-acetylglucosamine (6AzGlcNAc) can bypass the traditional GlcNAc-salvage pathway to generate uridine diphosphate sugar (UDP-6AzGlcNAc) (Chuh et al, 2014), resulting in labeling of O-GlcNAcylated proteins and suggesting that cellular metabolism is more accommodating to MCRs than previously appreciated While this phenomenon was confirmed and expanded by ourselves and other labs, a recent analysis of per-O-acetylated MCRs by the Wang and Chen labs showed that they can chemically react with cysteines on proteins when incubated with cell lysates at moderate to high concentrations (0.2–2.0 mM) (Qin et al, 2018; Hao et al, 2019), raising concerns about how much reporter-labeling is due to enzymatic glycosylation. The resulting protein mixture was subjected to the CuAAC conditions described below
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