Sialylation, a post-translational modification that impacts the structure, activity, and longevity of
glycoproteins has been thought to be controlled primarily by the expression of
sialyltransferases (STs). In this report we explore the complementary impact of metabolic flux on sialylation using a glycoengineering approach. Specifically, we treated three human breast cell lines (MCF10A, T-47D, and MDA-MB-231) with 1,3,4-O-Bu3ManNAc, a "high flux" metabolic precursor for the
sialic acid biosynthetic pathway. We then analyzed N-
glycan sialylation using solid phase extraction of
glycopeptides (SPEG) mass spectrometry-based proteomics under conditions that selectively captured
sialic acid-containing
glycopeptides, referred to as "sialoglycosites." Gene ontology (GO) analysis showed that flux-based changes to sialylation were broadly distributed across classes of
proteins in 1,3,4-O-Bu3ManNAc-treated cells. Only three categories of
proteins, however, were "highly responsive" to flux (defined as two or more sialylation changes of 10-fold or greater). Two of these categories were cell signaling and cell adhesion, which reflect well-known roles of
sialic acid in
oncogenesis. A third category-protein folding chaperones-was unexpected because little precedent exists for the role of glycosylation in the activity of these
proteins. The highly flux-responsive
proteins were all linked to
cancer but sometimes as
tumor suppressors, other times as proto-oncogenes, or sometimes both depending on sialylation status. A notable aspect of our analysis of metabolically glycoengineered breast cells was decreased sialylation of a subset of glycosites, which was unexpected because of the increased intracellular levels of sialometabolite "building blocks" in the 1,3,4-O-Bu3ManNAc-treated cells. Sites of decreased sialylation were minor in the MCF10A (<25% of all glycosites) and T-47D (<15%) cells but dominated in the MDA-MB-231 line (~60%) suggesting that excess
sialic acid could be detrimental in advanced
cancer and
cancer cells can evolve mechanisms to guard against hypersialylation. In summary, flux-driven changes to sialylation offer an intriguing and novel mechanism to switch between context-dependent pro- or anti-
cancer activities of the several
oncoproteins identified in this study. These findings illustrate how metabolic glycoengineering can uncover novel roles of
sialic acid in
oncogenesis.