Artificial Cysteine S‐Glycosylation Induced by Per‐O‐Acetylated Unnatural Monosaccharides during Metabolic Glycan Labeling

Abstract

The unexpected, non-enzymatic S-glycosylation of cysteine residues in various proteins by per-O-acetylated monosaccharides is described. This artificial S-glycosylation greatly compromises the specificity and validity of metabolic glycan labeling in living cells by per-O-acetylated azido and alkynyl sugars, which has been overlooked in the field for decades. It is demonstrated that the use of unacetylated unnatural sugars can avoid the artifact formation and a corrected list of O-GlcNAcylated proteins and O-GlcNAc sites in HeLa cells has been assembled by using N-azidoacetylgalactosamine (GalNAz).

Biological Functions and Analytical Strategies of Sialic Acids in Tumor

“…Amazingly, using an Ac 4 ManNAz metabolic labeling strategy, sialylated N-glycans on a select group of small noncoding RNAs (glycoRNAs), including Y RNAs, have been observed in mammalian cells [140], which may update our knowledge towards biochemistry, as we already know [141]. It is worth noting that per-O-acetylated monosaccharides can spontaneously react with numerous cysteines in proteomes with non-enzymatic catalysis, resulting in abnormal S-glycosylation [142]. In consideration of this condition, it is necessary to verify the results by specific glycosylation sites to exclude the S-glycosylation on cysteines when performing sialoglycoprotein research using Ac 4 ManNAz.…”

A Hierarchical Coding Strategy for Live Cell Imaging of Protein‐Specific Glycoform

“…The decoding of Sia code on G 1 with F‐ G 2 ‐Q (or Fuc code on G 3 with F′‐ G 4 ‐Q′) provides a hairpin opening‐derived fluorescence signal and releases P for the next catalytic cycle. A recent important finding of artificial cysteine glycosylation by the Chen group raises a caveat on the use of the metabolic engineering approach . In the case of MUC1, the three cysteines are located on the C‐terminal, intracellular region, and thus do not interfere with the extracellular HieCo imaging process.…”

Chemical and Biochemical Strategies To Explore the Substrate Recognition of O‐GlcNAc‐Cycling Enzymes

“…Ar ecent study also reportedn onspecific reactions of cysteine residues with acetylated GalNAc/GlcNAc analoguest hat have been widely used for metabolic labeling of O-GlcNAci nc ells. [23] Finally, imaging can be difficult with proteins that are not highly glycosylated, and distance limitations between the fluorophores might restrict the application of FRET on certainlarge substrate proteins.O ther strategies such as SNAP-tag, which exploits an alkyltransferase (O 6 -alkylguanine-DNA-alkyltransferase) to label fusion proteins with af luorophore, could also be adapted to investigate OGT substratepreferences in cells. [84]…”

“…Moreover, differential O‐GlcNAcylation could result from an equilibrium of OGT and OGA activities. A recent study also reported nonspecific reactions of cysteine residues with acetylated GalNAc/GlcNAc analogues that have been widely used for metabolic labeling of O ‐GlcNAc in cells . Finally, imaging can be difficult with proteins that are not highly glycosylated, and distance limitations between the fluorophores might restrict the application of FRET on certain large substrate proteins.…”

“…[11,12] Recent quantitative mass spectrometry studies have revealed that the O-GlcNAc level of certain proteinsc hanges in response to environmentals timuli and in different disease states. [13][14][15][16][17][18][19][20][21][22][23] Therefore, understanding what mechanisms regulate the enzyme activity and substrate specificity of OGT and OGA will help elucidate the biological functions of this modification in health and disease.…”