Robustness in glycosylation systems: effect of modified monosaccharides, acceptor decoys and azido sugars on cellular nucleotide-sugar levels and pattern of N-linked glycosylation.

ABSTRACT: Small molecule monosaccharide analogs (e.g. 4F-GlcNAc, 4F-GalNAc) and acceptor decoys (e.g. ONAP, SNAP) are commonly used as metabolic glycoengineering tools to perturb molecular and cellular recognition processes. Azido-derivatized sugars (e.g. ManNAz, GlcNAz, GalNAz) are also used as bioorthogonal probes to assay the glycosylation status of cells and tissue. With the goal of obtaining a systems-level understanding of how these compounds work, we cultured cells with these molecules and systematically evaluated their impact on: (i) cellular nucleotide-sugar levels, and (ii) N-linked glycosylation. To this end, we developed a streamlined, simple workflow to quantify nucleotide-sugar levels using amide-based hydrophilic interaction liquid chromatography (HILIC) separation followed by negative-mode electrospray ionization mass spectrometry (ESI-MS/MS) using an Orbitrap detector. N-Glycans released from cells were also procainamide functionalized and quantified using positive-mode ESI-MS/MS. Results show that all tested compounds changed the baseline nucleotide-sugar levels, with the effect being most pronounced for the fluoro-HexNAc compounds. These molecules depressed UDP-HexNAc levels in cells by up to 80%, while concomitantly elevating UDP-4F-GalNAc and UDP-4F-GlcNAc. While the measured changes in nucleotide-sugar concentration were substantial in many cases, their impact on N-linked glycosylation was relatively small. This may be due to the high nucleotide-sugar concentrations in the Golgi, which far exceed the K_M values of the glycosylating enzymes. Thus, the glycosylation system output exhibits 'robustness' even in the face of significant changes in cellular nucleotide-sugar concentrations.

SUBMITTER: Del Solar V 

PROVIDER: S-EPMC7423600 | biostudies-literature |

REPOSITORIES: biostudies-literature