Abstract
Sialic acid is a major determinant of carbohydrate-receptor interactions in many systems pertinent to human health and disease. N-Acetylmannosamine (ManNAc) is the first committed intermediate in the sialic acid biosynthetic pathway; thus, the mechanisms that control intracellular ManNAc levels are important regulators of sialic acid production. UDP-GlcNAc 2-epimerase and GlcNAc 2-epimerase are two enzymes capable of generating ManNAc from UDP-GlcNAc and GlcNAc, respectively. Whereas the former enzyme has been shown to direct metabolic flux toward sialic acid in vivo, the function of the latter enzyme is unclear. Here we study the effects of GlcNAc 2-epimerase expression on sialic acid production in cells. A key tool we developed for this study is a cell-permeable, small molecule inhibitor of GlcNAc 2-epimerase designed based on mechanistic principles. Our results indicate that, unlike UDP-GlcNAc 2-epimerase, which promotes biosynthesis of sialic acid, GlcNAc 2-epimerase can serve a catabolic role, diverting metabolic flux away from the sialic acid pathway.
Highlights
Introduction of compound3 into G2E* cells in the presence of Ac4ManLev resulted in a reversal of the phenotype attributed to GlcNAc 2-epimerase, indicating that the action of the enzyme was responsible for the low cell surface expression of unnatural sialic acids (Fig. 8)
We have previously found that the size of the N-acyl group on ManNAc derivatives can affect the efficiency of unnatural sialic acid biosynthesis in cells [37] and substrate activity with isolated enzymes in vitro
We were able to observe an effect on sialic acid biosynthesis by using unnatural metabolic substrates and by measurements of intracellular sialic acid levels
Summary
We study the effects of GlcNAc 2-epimerase expression on sialic acid production in cells. To investigate the function of GlcNAc 2-epimerase in cells, we identified a cell line lacking the enzyme, introduced the gene into these cells, and studied the effects of GlcNAc 2-epimerase expression on sialic acid biosynthetic flux. Guided by a proposed chemical mechanism [21], we prepared two novel substrate-based inhibitors of GlcNAc 2-epimerase and demonstrated that one of these inhibitors functions within cells to block the action of the enzyme. Using the inhibitor as a tool, we confirmed that the phenotype resulting from GlcNAc 2-epimerase expression was due to direct action of the enzyme Based on these data, we propose that GlcNAc 2-epimerase plays a catabolic role in sialic acid metabolism
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