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.

In this study, we investigated the use of metabolic oligosaccharide engineering and bio-orthogonal ligation reactions combined with lectin microarray and mass spectrometry to analyze sialoglycoproteins in the SW1990 human pancreatic cancer line. Specifically, cells were treated with the azido N-acetylmannosamine analog, 1,3,4-Bu3ManNAz, to label sialoglycoproteins with azide-modified sialic acids. The metabolically labeled sialoglyproteins were then biotinylated via the Staudinger ligation, and sialoglycopeptides containing azido-sialic acid glycans were immobilized to a solid support. The peptides linked to metabolically labeled sialylated glycans were then released from sialoglycopeptides and analyzed by mass spectrometry; in parallel, the glycans from azido-sialoglycoproteins were characterized by lectin microarrays. This method identified 75 unique N-glycosite-containing peptides from 55 different metabolically labeled sialoglycoproteins of which 42 were previously linked to cancer in the literature. A comparison of two of these glycoproteins, LAMP1 and ORP150, in histological tumor samples showed overexpression of these proteins in the cancerous tissue demonstrating that our approach constitutes a viable strategy to identify and discover sialoglycoproteins associated with cancer, which can serve as biomarkers for cancer diagnosis or targets for therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12014-015-9083-8) contains supplementary material, which is available to authorized users.

Metabolic oligosaccharide engineering is an emerging technology wherein non-natural monosaccharide analogs are exogenously supplied to living cells and are biosynthetically incorporated into cell surface glycans. A recently reported application of this methodology employs fluorinated analogs of ManNAc, GlcNAc and GalNAc to modulate selectin-mediated adhesion associated with leukocyte extravasation and cancer cell metastasis. This monograph outlines possible mechanisms underlying the altered adhesion observed in analog-treated cells; these range from the most straightforward explanation (e.g., structural changes to the selectin ligands ablate interaction with their receptors) to the alternative mechanism where the analogs inhibit or otherwise perturb ligand production to more indirect mechanisms (e.g., changes to the biophysical properties of the selectin binding partner, the nanoenviroment of the binding partners, or the entire cell surface).

Evidence for photo-induced radical disulfide bond scrambling in the gas phase during matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is described. The phenomenon was observed during the analysis of tryptic peptides from insulin and was confirmed in the determination of disulfide bonds in the rhamnose-binding lectin SEL24K from the Chinook salmon Oncorhynchus tshawytscha. A possible mechanism for this surprising scrambling is proposed. Despite this finding, the disulfide bond pattern in SEL24K was assigned unambiguously by a multi-enzyme digestion strategy in combination with MALDI mass spectrometry. The pattern was found to be symmetrical in the tandem repeat sequence of SEL24K. To the best of our knowledge, this is the first report of disulfide bond scrambling in the gas phase during MALDI-MS analysis. This observation has important ramifications for unambiguous assignment of disulfide bonds. (J Am Soc

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Structural glycobiology has traditionally been a challenging field due to a limited set of tools available to investigate the diverse and complex glycan molecules. However, we cannot ignore that glycans play critical roles in health as well as in disease, and are present in more than 50% of all proteins and on over 80% of all surface proteins. Chemoenzymatic glycoengineering (CGE) methods are a powerful set of tools to synthesize complex glycans, but the full potential of these methods have not been explored in cell biology yet. Herein, we report the labeling of live Chinese hamster ovary (CHO) cells by employing three highly specific glycosyltransferases: a sialyltransferase, a galactosyltransferase, and an N-acetyl-glucosaminyl transferase. We verified our results by bio-orthogonal blots and further rationalized them by computational modeling. We expect CGE applications in cell biology to rise and their implementation will assist in structural-functional discoveries in glycobiology. This research will contribute to this effort.

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