Synthesis of <i>N</i>-acetylneuraminic acid and of CMP-<i>N</i>-acetylneuraminic acid in the rat liver cell

Ferwerda, W; Blok, Corrie M.; Rinsum, J Van

doi:10.1042/bj2160087

Cited by 18 publications

(8 citation statements)

References 26 publications

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“…For CMP-NeuAc comparable values have been found with colorimetric quantification [13,25]. The values reported for CDP-choline, determined enzymically [26], and cytidine phosphates obtained with the h.p.l.c.…”

Section: Discussionsupporting

confidence: 55%

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Pyrimidine metabolism and sugar nucleotide synthesis in rat liver

Rijcken

Hooghwinkel

Ferwerda

1990

Biochemical Journal

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With radioactive precursors, the labelling kinetics of the soluble pyrimidine nucleotides and of RNA were measured in rat liver to determine the contribution of the metabolic flows through synthesis de novo and the salvage pathway. To separate and quantify all pyrimidine nucleotides, an h.p.l.c. technique was developed using anion-exchange chromatography and reversed-phase chromatography. The concentrations of cytidine nucleotides were in the range of 30-45 nmol/g wet weight, and the concentrations of the uridine phosphates and of the UDP-sugars were approx. 6 and 20 times higher respectively. After a single injection of [14C]orotic acid and of [3H]cytidine, the specific radioactivities were determined as a function of time. The 1'C/3H ratio was calculated and gave a good indication of the involvement of the different flows. It could be concluded that UTP derived from synthesis de novo and from the salvage pathway is not completely mixed before being utilized. The flow of the salvage pathway is relatively more directed to RNA synthesis in the nucleus and that of synthesis de novo to cytoplasmic processes. For CTP it could also be concluded that the flow of the salvage pathway was relatively more directed to RNA synthesis in the nucleus. Because of the nuclear localization of the enzyme CMP-NeuAc (N-acetylneuraminate) synthase, special attention was paid to CMP-NeuAc. However, a conclusion about a location about the synthesis of CMP-NeuAc could not unequivocally be drawn, because of the small differences in 14C/3H ratio and the different values for the CDP-lipids.

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Section: Discussionsupporting

confidence: 55%

“…Male Wistar rats of age 2-3 months (weighing 150-200 g) were kept on a light/dark schedule and fed for 2 h each day as described previously [13]. The rats were starved for 24 h before use.…”

Section: Animalsmentioning

confidence: 99%

Pyrimidine metabolism and sugar nucleotide synthesis in rat liver

Rijcken

Hooghwinkel

Ferwerda

1990

Biochemical Journal

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“…This seems to be in contrast to observations that [ 3 H]NeuAc is a rather inefficient substrate for labeling sialoglycoconjugates radioactively as compared to [ 3 H]ManNAc [20]. It has been suggested that the cytosolic N ‐acetylneuraminic acid 9‐phosphate phosphatase, the last enzyme involved in NeuAc biosynthesis, might also play a role in recruiting NeuAc to the nucleus where CMP‐activation occurs [46]. In contrast to ManNAc‐derived NeuAc‐9‐phosphate, directly added unphosphorylated NeuAc is not processed by this enzyme and might therefore reach the nucleus less efficiently.…”

Section: Discussionmentioning

confidence: 74%

Evidence for efficient uptake and incorporation of sialic acid by eukaryotic cells

Oetke

Hinderlich

Brossmer

et al. 2001

European Journal of Biochemistry

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Sialic acids are the most abundant terminal carbohydrate moiety on cell surface glycoconjugates in eukaryotic cells and are of functional importance for many biological ligand-receptor interactions. It is a widely accepted view that sialic acids cannot be efficiently taken up from the extracellular space by eukaryotic cells. To test this assumption, we cultivated two recently identified human hematopoetic cell lines which are hyposialylated due to a deficiency in de novo sialic acid biosynthesis in the presence of N-acetylneuraminic acid (NeuAc), the most frequently found sialic acid. Surprisingly, NeuAc medium supplementation rapidly and potently compensated for the endogenous hyposialylation in a concentration-dependent manner, resulting in the presentation of cell surface sialoglycans involved in cell adhesion, virus infection and signal transduction. We provide several lines of experimental evidence that all suggest that NeuAc was neither extracellularly incorporated nor degraded to a less complex sugar before uptake. Importantly, NeuAc induced a marked increase in intracellular CMP-NeuAc levels in both human cell lines and in primary cells regardless of the prior sialylation status of the cells. Studies employing 9-[ 3 H]NeuAc revealed an uptake consistent with the observed incorporation of unlabeled NeuAc. We propose the existence of an efficient uptake mechanism for NeuAc in eukaryotic cells.

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“…Consequently, the specific productivity which is later an input parameter for the glycosylation model, was described with a Gaussian distribution with optimal culture pH of 7.15 (experimentally determined). The nucleotide sugars uridine diphospate N‐acetylglucosamine, (UDP‐GlcNAc), guanosine diphosphate fucose (GDP‐Fuc), and uridine diphospate galactose (UDP‐Gal) are produced in the cytosol whereas cytidine diphosphate N‐acetylneuraminic acid (CMP‐Neu5Ac) is synthesized in the nucleus . The nucleotide sugar precursors are then transferred to the ER and Golgi apparatus via active antitransporters.…”

Section: Methodsmentioning

confidence: 99%

Controlling the time evolution of mAb N‐linked glycosylation ‐ Part II: Model‐based predictions

Villiger

Scibona

Stettler

et al. 2016

Biotechnology Progress

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N-linked glycosylation is known to be a crucial factor for the therapeutic efficacy and safety of monoclonal antibodies (mAbs) and many other glycoproteins. The nontemplate process of glycosylation is influenced by external factors which have to be tightly controlled during the manufacturing process. In order to describe and predict mAb N-linked glycosylation patterns in a CHO-S cell fed-batch process, an existing dynamic mathematical model has been refined and coupled to an unstructured metabolic model. High-throughput cell culture experiments carried out in miniaturized bioreactors in combination with intracellular measurements of nucleotide sugars were used to tune the parameter configuration of the coupled models as a function of extracellular pH, manganese and galactose addition. The proposed modeling framework is able to predict the time evolution of N-linked glycosylation patterns during a fed-batch process as a function of time as well as the manipulated variables. A constant and varying mAb N-linked glycosylation pattern throughout the culture were chosen to demonstrate the predictive capability of the modeling framework, which is able to quantify the interconnected influence of media components and cell culture conditions. Such a model-based evaluation of feeding regimes using high-throughput tools and mathematical models gives rise to a more rational way to control and design cell culture processes with defined glycosylation patterns. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1135-1148, 2016.

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Synthesis of N-acetylneuraminic acid and of CMP-N-acetylneuraminic acid in the rat liver cell

Cited by 18 publications

References 26 publications

Pyrimidine metabolism and sugar nucleotide synthesis in rat liver

Pyrimidine metabolism and sugar nucleotide synthesis in rat liver

Evidence for efficient uptake and incorporation of sialic acid by eukaryotic cells

Controlling the time evolution of mAb N‐linked glycosylation ‐ Part II: Model‐based predictions

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