This study reports a global glycoproteomic analysis of pancreatic cancer cells that describes how flux through the sialic acid biosynthetic pathway selectively modulates a subset of N-glycosylation sites found within cellular proteins. These results provide evidence that sialoglycoprotein patterns are not determined exclusively by the transcription of biosynthetic enzymes or the availability of N-glycan sequons; instead, bulk metabolic flux through the sialic acid pathway has a remarkable ability to increase the abundance of certain sialoglycoproteins while having a minimal impact on others. Specifically, of 82 glycoproteins identified through a mass spectrometry and bioinformatics approach, ϳ31% showed no change in sialylation, ϳ29% exhibited a modest increase, whereas ϳ40% experienced an increase of greater than twofold. The surfaces of mammalian cells are covered with a dense layer of carbohydrates, collectively known as the glycocalyx, that influence many aspects of the interaction between a cell and its microenvironment. To date, the biosynthesis of cell surface displayed glycans has been thought to be controlled largely by individual glycosyltransferases based on the assumption that flux through the metabolic pathways that supply activated nucleotide sugar donors (the substrates for these enzymes) is not a limiting factor. For example, this premise has been used in mathematical models of sialylation (1), where concentrations of CMP-Neu5Ac in the lumen of the Golgi were assumed to be much higher than the K m of sialyltransferases (2). In the past several years, the idea that nucleotide sugars, exemplified by CMP-Neu5Ac (shown in Fig. 1A), are not a limiting or controlling factor in glycosylation has garnered one major and unambiguous exception, notably that changes in flux through the hexosamine biosynthetic pathway (HBP) 1 can alter UDP-GlcNAc levels with profound consequences on the branching of N-linked glycans (3). By changing the valence of these glycoconjugates, flux through the HBP can alter the galectin lattice and affect a host of downstream biological events including cancer progression (4); cell differentiation and proliferation (3); and autoimmunity, metabolic syndromes, and aging (5).In this report, we demonstrate that the HBP is not unique in its ability to control surface glycoproteins via bulk metabolic flux. In particular, counter to earlier assumptions that flux through the sialic acid pathway does not significantly alter the sialylation of individual glycans (2), analysis of two "high-demand" sialoglycans (i.e. polysialylated NCAM (6) and podocalyxin (7)) suggested that fluctuations in the intracellular concentrations of sialic acid and the corresponding supply of CMP-Neu5Ac critically affected their production. In the current report, we used a global cell level approach to investigate whether these two examples were outliers or whether metabolic flux controls the surface display of sialic acid with fine resolution across a wide range of N-linked glycoproteins. We found that the sialylat...

Metabolic oligosaccharide engineering is a maturing technology capable of modifying cell surface sugars in living cells and animals through the biosynthetic installation of non-natural monosaccharides into the glycocalyx. A particularly robust area of investigation involves the incorporation of azide functional groups onto the cell surface, which can then be further derivatized using “click chemistry.” While considerable effort has gone into optimizing the reagents used for the azide ligation reactions, less optimization of the monosaccharide analogues used in the preceding metabolic incorporation steps has been done. This study fills this void by reporting novel butanoylated ManNAc analogues that are used by cells with greater efficiency and less cytotoxicity than the current “gold standard,” which are peracetylated compounds such as Ac4ManNAz. In particular, tributanoylated, N-acetyl, N-azido, and N-levulinoyl ManNAc analogues with the high flux 1,3,4-O-hydroxyl pattern of butanoylation were compared with their counterparts having the pro-apoptotic 3,4,6-O-butanoylation pattern. The results reveal that the ketone-bearing N-levulinoyl analogue 3,4,6-O-Bu3ManNLev is highly apoptotic, and thus is a promising anti-cancer drug candidate. By contrast, the azide-bearing analogue 1,3,4-O-Bu3ManNAz effectively labeled cellular sialoglycans at concentrations ∼3 to 5-fold lower (e.g., at 12.5 to 25 μM) than Ac4ManNAz (50 to 150 μM) and exhibited no indications of apoptosis even at concentrations up to 400 μM. In summary, this work extends emerging structure activity relationships that predict the effects of short chain fatty acid modified monosaccharides on mammalian cells and also provides a tangible advance in efforts to make metabolic oligosaccharide engineering a practical technology for the medical and biotechnology communities.

Background: Delta-like ligand 4 (Dll4) is a Notch ligand that is upregulated by hypoxia and vascular endothelial growth factor-A (VEGF-A) and is reported to have a role in tumor angiogenesis. Evidence from xenograft studies suggests that inhibiting Dll4–Notch signalling may overcome resistance to anti-VEGF therapy. The aim of this study was to characterise the expression of Dll4 in colon cancer and to assess whether it is associated with markers of hypoxia and prognosis. Method: In all, 177 colon cancers were represented in tissue microarrays. Immunohistochemistry was performed using validated antibodies against Dll4, VEGF, hypoxia-inducible factor (HIF)-1 α , HIF-2 α , prolyl hydroxylase (PHD)1, PHD2, PHD3 and carbonic anhydrase 9 (CA9). Results: The expression of Dll4 was observed preferentially in the endothelium of 71% (125 out of 175) of colon cancers, but not in the endothelium adjacent to normal mucosa (none out of 107, P <0.0001). The expression of VEGF was significantly associated with HIF-2 α ( P <0.0001) and Dll4 ( P =0.010). Only HIF-2 α had a significant multivariate prognostic effect (hazard ratio 1.61, 95% confidence interval 1.01–2.57). Delta-like ligand 4 was also expressed by neoplastic cells, particularly neoplastic goblet cells. Conclusion: Endothelial expression of Dll4 is not a prognostic factor, but is significantly associated with VEGF. Assessing endothelial Dll4 expression may be critical in predicting response to anti-VEGF therapies.