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Figure 5 | Genome Biology

Figure 5

From: Characterizing glycosylation pathways

Figure 5

Strategy for metabolic cell selection for defects in the sialic acid pathway. The sialic acid pathway beginning with UDP-GlcNAc proceeds by the sequential action of (a,b) UDP-GlcNAc 2-epimerase/ManNAc 6-kinase; (c) sialic acid 9-phosphate synthase; (d) sialic acid 9-phosphatase; (e) CMP-sialic acid synthetase; (f) CMP-sialic acid Golgi transporter; and (g) sialyltransferase (several in human); to install a sialic acid on glycoconjugates destined for the cell surface. The substrate-based cell selection approach begins with (1) the interception of the pathway with ManLev, an unnatural analog of ManNAc. The pathway converts this unnatural substrate into glycan-bound SiaLev containing a selectable marker, the ketone (2). The ketone can be selectively labeled with biotin hydrazide and the cells stained with FITC-labeled avidin (3). Sorting of low-SiaLev cells (shown, or high-SiaLev cells, not shown) by flow cytometry (4) allows for the high-throughput selection of cells harboring rare metabolic mutations. After sorting, cells are allowed to recover and then the sorted cell population is analyzed for ketone expression (5). These five steps are repeated iteratively until the desired phenotypic endpoint of high- or low-SiaLev expression is obtained.

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