N-Glycomic Analysis of the Cell Shows Specific Effects of Glycosyl Transferase Inhibitors

Abstract

Glycomic profiling methods were used to determine the effect of metabolic inhibitors on glycan production. These inhibitors are commonly used to alter the cell surface glycosylation. However, structural analysis of the released glycans has been limited. In this research, the cell membranes were enriched and the glycans were released to obtain the N-glycans of the glycocalyx. Glycomic analysis using liquid chromatography–mass spectrometry (LC–MS) with a PGC chip column was used to profile the structures in the cell membrane. Glycans of untreated cells were compared to glycans of cells treated with inhibitors, including kifunensine, which inhibits the formation of complex- and hybrid-type structures, 2,4,7,8,9-Penta-O-acetyl-N-acetyl-3-fluoro-b-d-neuraminic acid methyl ester for sialylated glycans, 2-deoxy-2-fluorofucose, and 6-alkynyl fucose for fucosylated glycans. Kifunensine was the most effective, converting nearly 95% of glycans to high mannose types. The compound 6-alkynyl fucose inhibited some fucosylation but also incorporated into the glycan structure. Proteomic analysis of the enriched membrane for the four inhibitors showed only small changes in the proteome accompanied by large changes in the N-glycome for Caco-2. Future works may use these inhibitors to study the cellular behavior associated with the alteration of glycosylation in various biological systems, e.g., viral and bacterial infection, drug binding, and cell–cell interactions.

Host Cell Glycocalyx Remodeling Reveals SARS-CoV-2 Spike Protein Glycomic Binding Sites

“…Compositional profiles were generated for the modified cells, using the sum of the intensities for similar glycan types from the LC-MS analysis. These inhibitors have recently been applied for altering cell surface glycosylation to yield similar results ( Zhou et al, 2021 ) ( Figure 4B ). 2F-Fucose inhibits fucosylation by being converted to the sugar nucleotide GDP-2F-Fuc (( Villalobos et al, 2015 )).…”

“…Molecular Biosciences | www.frontiersin.org March 2022 | Volume 9 | Article 799703recently been applied for altering cell surface glycosylation to yield similar results(Zhou et al, 2021) (Figure4B). 2F-Fucose inhibits fucosylation by being converted to the sugar nucleotide GDP-2F-Fuc ((Villalobos et al, 2015)).…”

Small molecule inhibitors of mammalian glycosylation

“…When compared to other inhibitors like 2FF, 6AF seems to have similar inhibition potential, although results vary in different cell lines and experimental conditions. For example, 6AF is reported to be more potent than 2FF in MEF, [240] Caco-2, and PNT2 cell lines, while 2FF performs better in A549 [241] . An additional complexity emerges from the analysis of the post-inhibition glycome, revealing that alkynylated fucose analogues are incorporated in the cell [241] .…”

“…For example, 6AF is reported to be more potent than 2FF in MEF, [240] Caco-2, and PNT2 cell lines, while 2FF performs better in A549 [241] . An additional complexity emerges from the analysis of the post-inhibition glycome, revealing that alkynylated fucose analogues are incorporated in the cell [241] .…”

The role of FUT8‐catalyzed core fucosylation in Alzheimer's amyloid‐β oligomer‐induced activation of human microglia

“…As expected, 2FF and 6AF had no impact on the FUT8 protein level (Figure 4b), indicating that their effects were not mediated by FUT8 downregulation. As a negative control, 2,4,7,8,9-Penta-O-acetyl-N-acetyl-3-fluoro-b-D-neuraminic acid methyl ester (3FS), a sialylation inhibitor (Zhou et al, 2021), did not reduce fucosylation (Figure 4a).…”