Dynamic tracing of sugar metabolism reveals the mechanisms of action of synthetic sugar analogs

Scherpenzeel, Monique van; Conte, Federica; Büll, Christian; Ashikov, Angel; Hermans, Esther; Willems, Anke P.; Tol, Walinka van; Kragt, Else; Noga, Marek; Moret, Ed E.; Heise, Torben; Langereis, Jeroen D.; Rossing, Emiel; Zimmermann, Michael; Rubio‐Gozalbo, M. Estela; Jonge, Marien I. de; Adema, Gosse J.; Zamboni, Nicola; Boltje, Thomas J.; Lefeber, Dirk

doi:10.1093/glycob/cwab106

Cited by 19 publications

(37 citation statements)

References 51 publications

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“…Firstly, we analyzed the production of CMP-Sia in wildtype flies and CSAS mutants by a liquid chromatography-mass spectrometry approaches (see Materials and Methods). A prominent peak corresponding to CMP-Sia was detected in wildtype flies as shown before (Scherpenzeel et al, 2021), while no CMP-Sia was found in CSAS mutants. Transgenic rescue using UAS-GAL4 system resulted in the restoration of CMP-Sia biosynthesis in the mutants (Fig 4A-B).…”

Section: Resultssupporting

confidence: 76%

Glia-neuron coupling via a bipartite sialylation pathway promotes neural transmission and stress tolerance in Drosophila

Scott

Novikov

Ugur

et al. 2022

Preprint

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Modification by sialylated glycans can affect protein functions, underlying mechanisms that control animal development and physiology. Sialylation relies on a dedicated pathway involving evolutionarily conserved enzymes, including CMP-sialic acid synthetase (CSAS) and sialyltransferase (SiaT) that mediate the activation of sialic acid and its transfer onto glycan termini, respectively. In Drosophila, CSAS and DSiaT genes function in the nervous system, affecting neural transmission and excitability. We found that these genes function in different cells: the function of CSAS is restricted to glia, while DSiaT functions in neurons. This partition of the sialylation pathway allows for regulation of neural functions via a glia-mediated control of neural sialylation. The sialylation genes were shown to be required for tolerance to heat and oxidative stress and for maintenance of the normal level of voltage-gated sodium channels. Our results uncovered a unique bipartite sialylation pathway that mediates glia-neuron coupling and regulates neural excitability and stress tolerance.

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Section: Resultssupporting

confidence: 76%

Glia-neuron coupling via a bipartite sialylation pathway promotes neural transmission and stress tolerance in Drosophila

Scott

Novikov

Ugur

et al. 2022

Preprint

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“…4, with details in Fig. S5) [41]. The overexpression of ST6GAL1 in hCK WT and hCK-B3GNT2 cells compared to MDCK WT cells resulted in elevated levels of sialylation and thereby lower levels of available CMP-Neu5Ac, which was also demonstrated in the sugar nucleotide analysis.…”

Section: Resultsmentioning

confidence: 85%

“…The supernatant was taken and dried in the vacuum concentrator. Samples were frozen at −80°C until analysis using an ion-pair UHPLC-QqQ 1290-6490 Agilent mass spectrometer by Glycomscan BV (Oss, the Netherlands) [41].…”

Section: Methodsmentioning

confidence: 99%

Contemporary human H3N2 influenza A viruses require a low threshold of suitable glycan receptors for efficient infection

Spruit

Sweet

Bestebroer

et al. 2022

Preprint

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Recent human H3N2 influenza A viruses (IAV) have evolved to employ elongated glycans terminating in α2,6-linked sialic acid as their receptors. These glycans are displayed in low abundancies by cells commonly employed to propagate these viruses (MDCK and hCK), resulting in low or no viral propagation. Here, we examined whether the overexpression of the glycosyltransferases B3GNT2 and B4GALT1, which are responsible for the elongation of poly-N-acetyllactosamines (LacNAc), would result in improved A/H3N2 propagation. Stable overexpression of B3GNT2 and B4GALT1 in MDCK and hCK cells was achieved by lentiviral integration and subsequent antibiotic selection and confirmed by qPCR and protein mass spectrometry experiments. Flow cytometry and glycan mass spectrometry experiments using the B3GNT2 and/or B4GALT1 knock-in cells demonstrated increased binding of viral hemagglutinins and the presence of a larger number of LacNAc repeating units, especially on hCK-B3GNT2 cells. An increase in the number of glycan receptors did, however, not result in a greater infection efficiency of recent human H3N2 viruses. Based on these results, we propose that H3N2 IAVs require a low number of suitable glycan receptors to infect cells and that an increase in the glycan receptor display above this threshold does not result in improved infection efficiency.

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“…Another hypothesis regarding the diffuse staining of labeled GCs into cells is supported by a recent study showing that incubation of human fibroblasts with Ac 4 ManNAl (Ac 4 ManNPoc) resulted in biosynthesis of not only CMP-SiaNAl, but also UDP-HexNAl 42 . The latter may come from the hijacking of unnatural ManNAc by GlcNAc-2-epimerase (RENBP), leading to the biosynthesis of chemically modified UDP-GlcNAc through the salvage pathway 43 (Fig.…”

Section: Discussionmentioning

confidence: 95%

Switching azide and alkyne tags on bioorthogonal reporters in metabolic labeling of sialylated glycoconjugates: a comparative study

Scache

Rigolot

Lion

et al. 2022

Sci Rep

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Sialylation of cell surface glycans plays an essential role in cell–cell interaction and communication of cells with their microenvironment. Among the tools that have been developed for the study of sialylation in living cells, metabolic oligosaccharide engineering (MOE) exploits the biosynthetic pathway of sialic acid (Sia) to incorporate unnatural monosaccharides into nascent sialylatedglycoconjugates, followed by their detection by a bioorthogonal ligation of a molecular probe. Among bioorthogonal reactions, the copper-catalyzed azide-alkyne cycloaddition (CuAAC) is the only ligation where both reactive tags can be switched on the chemical reporter or on the probe, making this reaction very flexible and adaptable to various labeling strategies. Azide- and alkyne-modified ManNAc and Sia reporters have been widely used, but per-O-acetylated ManNAz (Ac4ManNAz) remains the most popular choice so far for tracking intracellular processing of sialoglycans and cell surface sialylation in various cells. Taking advantage of CuAAC, we compared the metabolic incorporation of ManNAl, ManNAz, SiaNAl, SiaNAz and Ac4ManNAz in the human colon cell lines CCD841CoN, HT29 and HCT116, and in the two gold standard cell lines, HEK293 and HeLa. Using complementary approaches, we showed marked differences in the efficiency of labeling of sialoglycoproteins between the different chemical reporters in a given cell line, and that switching the azide and alkyne bioorthogonal tags on the analogs highly impacted their metabolic incorporation in the human colon cell lines. Our results also indicated that ManNAz was the most promiscuous metabolized reporter to study sialylation in these cells.

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Dynamic tracing of sugar metabolism reveals the mechanisms of action of synthetic sugar analogs

Cited by 19 publications

References 51 publications

Glia-neuron coupling via a bipartite sialylation pathway promotes neural transmission and stress tolerance in Drosophila

Glia-neuron coupling via a bipartite sialylation pathway promotes neural transmission and stress tolerance in Drosophila

Contemporary human H3N2 influenza A viruses require a low threshold of suitable glycan receptors for efficient infection

Switching azide and alkyne tags on bioorthogonal reporters in metabolic labeling of sialylated glycoconjugates: a comparative study

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