Molecular outcomes of ManLev-based selection. Three mutational outcomes of metabolic selection include (1, red) a defect in UDP-GlcNAc 2-epimerase (a) that renders this enzyme refractory to feedback inhibition by CMP-Sia. The consequent increase in endogenous ManNAc production competitively excludes the unnatural substrate ManLev from the pathway, accounting for the 'low-SiaLev' phenotype. The continued flux of natural substrate through the pathway also explains the normal cell-surface glycan expression of these mutant cells. Another possible mutational outcome of a low-SiaLev selection is a defect in the sialic acid synthase (2, green). Like the mutant epimerase cells, these cells also have normal cell-surface glycan expression, with the internal defect being 'masked' by acquisition of exogenous Sia downstream of the defect. Finally, a 'high-SiaLev' phenotype results from neo-expression of poly-Sia (3, blue), as a result of upregulation of NCAM. Unlike the first two cell lines, these cells have aberrant cell-surface carbohydrate presentation, having gained expression of a carbohydrate epitope characteristic of highly metastatic cancer (Enzymes a through gare identified in the legend to Figure 5).