RESUMEN
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).
Asunto(s)
Cisteína/química , Monosacáridos/química , Acetilación , Alquinos/química , Azidas/química , Biotina/química , Biotina/metabolismo , Cisteína/metabolismo , Glutatión/química , Glicosilación , Células HeLa , Humanos , Ingeniería Metabólica , Monosacáridos/metabolismo , Péptidos/análisis , Espectrometría de Masas en TándemRESUMEN
We herein report a chemical decaging strategy for the inâ situ generation of neuramic acid (Neu), a unique type of sialic acid, on live cells by the use of a palladium-mediated bioorthogonal elimination reaction. Palladium nanoparticles (Pdâ NPs) were found to be a highly efficient and biocompatible depropargylation catalyst for the direct conversion of metabolically incorporated N-(propargyloxycarbonyl)neuramic acid (Neu5Proc) into Neu on cell-surface glycans. This conversion chemically mimics the enzymatic de-N-acetylation of N-acetylneuramic acid (Neu5Ac), a proposed mechanism for the natural occurrence of Neu on cell-surface glycans. The bioorthogonal elimination was also exploited for the manipulation of cell-surface charge by unmasking the free amine at C5 to neutralize the negatively charged carboxyl group at C1 of sialic acids.
Asunto(s)
Nanopartículas del Metal/química , Ácidos Neuramínicos/síntesis química , Paladio/química , Paladio/farmacología , Polisacáridos/metabolismo , Ácidos Siálicos/química , Ácidos Siálicos/síntesis química , Acetilación , Animales , Células CHO , Catálisis , Supervivencia Celular/efectos de los fármacos , Química Clic , Cricetulus , Citometría de Flujo , Humanos , Células Jurkat , Ácidos Neuramínicos/química , Ácidos Neuramínicos/metabolismo , Polisacáridos/química , Ácidos Siálicos/biosíntesis , Propiedades de SuperficieRESUMEN
Sialic acid analogues containing a unique chemical functionality or chemical reporter have been metabolically incorporated into sialylated glycans. This process, termed metabolic glycan labeling, has emerged as a powerful tool for studying sialylation as well as other types of glycosylation. Currently, this technique can install only a single functionality. Here we describe a strategy for dual labeling of sialylated glycans using a new class of bifunctional sialic acid analogues containing two distinct chemical reporters at the N-acyl and C9 positions. These bifunctional unnatural sialic acids were metabolically incorporated into cellular glycans, where the two chemical reporters exerted their distinct functions. This approach expands the capability of metabolic glycan labeling to probe sialylation and glycan-protein interactions.
