Defects in Galactose Metabolism and Glycoconjugate Biosynthesis in a UDP-Glucose Pyrophosphorylase-Deficient Cell Line Are Reversed by Adding Galactose to the Growth Medium

Durrant, Christelle; Fuehring, J.I.; Willemetz, Alexandra; Chrétien, Dominique; Sala, G; Ghidoni, Riccardo; Katz, Abram; Rötig, Agnès; Thelestam, Mónica; Ermonval, Myriam; Moore, Stuart

doi:10.3390/ijms21062028

Cited by 8 publications

(3 citation statements)

References 59 publications

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“…UDP-glucose and UPD-galactose are utilized in many biosynthetic pathways, including nucleotide and galactose metabolism. However, despite abnormalities in the nucleotide-activated sugars UDP-glucose and UDP-galactose, there were no indications of misdirected N-glycosylation (Figure S4) [27]. In general, metabolome analysis of dried blood spots revealed that PPA1 deficiency can not only lead to the derangement of the galactose metabolism and PPi formation but also broader abnormalities in amino acid, fatty acid and nucleotide metabolism.…”

Section: Discussionmentioning

confidence: 88%

PPA1 Deficiency Causes a Deranged Galactose Metabolism Recognizable in Neonatal Screening

Achleitner,

Jans,

Ebner

et al. 2023

Metabolites

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Two siblings showed increased galactose and galactose-related metabolites in neonatal screening. Diagnostic workup did not reveal abnormalities in any of the known disease-causing enzymes involved in galactose metabolism. Using whole-exome sequencing, we identified a homozygous missense variant in PPA1 encoding the cytosolic pyrophosphatase 1 (PPA1), c.557C>T (p.Thr186Ile). The enzyme activity of PPA1 was determined using a colorimetric assay, and the protein content was visualized via western blotting in skin fibroblasts from one of the affected individuals. The galactolytic activity of the affected fibroblasts was determined by measuring extracellular acidification with a Seahorse XFe96 analyzer. PPA1 activity decreased to 22% of that of controls in the cytosolic fraction of homogenates from patient fibroblasts. PPA1 protein content decreased by 50% according to western blot analysis, indicating a reduced stability of the variant protein. The extracellular acidification rate was reduced in patient fibroblasts when galactose was used as a substrate. Untargeted metabolomics of blood samples revealed an elevation of other metabolites related to pyrophosphate metabolism. Besides hyperbilirubinemia in the neonatal period in one child, both children were clinically unremarkable at the ages of 3 and 14 years, respectively. We hypothesize that the observed metabolic derangement is a possible mild manifestation of PPA1 deficiency. Unresolved abnormalities in galactosemia screening might result in the identification of more individuals with PPA1 deficiency, a newly discovered inborn metabolic disorder (IMD).

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Section: Discussionmentioning

confidence: 88%

PPA1 Deficiency Causes a Deranged Galactose Metabolism Recognizable in Neonatal Screening

Achleitner,

Jans,

Ebner

et al. 2023

Metabolites

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“…The decrease in l ‐arabitol content reduces the accumulation of cytoprotective substances, resulting in a decrease in the content of synthetic polysaccharides (Zhou et al, 2018). Galactose metabolism provides the precursor uridine diphosphate (UDPG) for polysaccharide synthesis (Durrant et al, 2020). The d‐ mannitol content in the MS group was higher than that in the OS group.…”

Section: Resultsmentioning

confidence: 99%

Poly‐pathways metabolomics for high‐yielding cordycepin of Cordyceps militaris

Sun

Wang

et al. 2022

Biomedical Chromatography

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Cordycepin is an important quality control marker in Cordyceps militaris. This study aimed to explain the metabolic mechanisms for high‐yielding cordycepin of C. militaris. In this study, high‐yielding strains of cordycepin were obtained by ultraviolet mutagenesis, and the polysaccharide and protein contents were also changed. In high‐yielding strains, the protein content significantly increased, whereas the polysaccharide content decreased. Simultaneously, metabolic differences for high‐ and low‐yielding cordycepin strains were detected by metabolomics. Metabolomics results showed that the relative content of most metabolites decreased in high‐yielding cordycepin strains. Various metabolic pathways have been altered in high‐yielding cordycepin strains, such as the citric acid cycle, purine metabolism, and pyrimidine metabolism, leading to an increase in cordycepin content. In addition, changes in metabolic poly‐pathways related to polysaccharide and protein synthesis, such as galactose metabolism and amino acid metabolism, promoted an increase in cordycepin content. This study analyzes the high yield of cordycepin in C. militaris at the metabolic level and provides a theoretical basis for further increasing cordycepin content.

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“…This is a significant advantage over traditional MS methods, which cannot differentiate between isobaric compounds. Using HRMS, the glucose and galactose were reported to have a significant impact on CHO cell metabolism, while other sugars (such as GDP-Fucose, UDP-Glc/UDP-Gal, UDP-GlcNAc/UDP-GalNAc) had no or minor effect ( Durrant et al, 2020 ). HRMS can also be utilized to analyse glycosaminoglycans (GAGs), which are complex carbohydrates that play a critical role in various biological processes ( Torres CL.…”

Section: Evolving Methods and Future Perspectives In Cho Metabolomicsmentioning

confidence: 99%

Advancements in CHO metabolomics: techniques, current state and evolving methodologies

Singh,

Fatima,

Thakur

et al. 2024

Front. Bioeng. Biotechnol.

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Background: Investigating the metabolic behaviour of different cellular phenotypes, i.e., good/bad grower and/or producer, in production culture is important to identify the key metabolite(s)/pathway(s) that regulate cell growth and/or recombinant protein production to improve the overall yield. Currently, LC-MS, GC-MS and NMR are the most used and advanced technologies for investigating the metabolome. Although contributed significantly in the domain, each technique has its own biasness towards specific metabolites or class of metabolites due to various reasons including variability in the concept of working, sample preparation, metabolite-extraction methods, metabolite identification tools, and databases. As a result, the application of appropriate analytical technique(s) is very critical.Purpose and scope: This review provides a state-of-the-art technological insights and overview of metabolic mechanisms involved in regulation of cell growth and/or recombinant protein production for improving yield from CHO cultures.Summary and conclusion: In this review, the advancements in CHO metabolomics over the last 10 years are traced based on a bibliometric analysis of previous publications and discussed. With the technical advancement in the domain of LC-MS, GC-MS and NMR, metabolites of glycolytic and nucleotide biosynthesis pathway (glucose, fructose, pyruvate and phenylalanine, threonine, tryptophan, arginine, valine, asparagine, and serine, etc.) were observed to be upregulated in exponential-phase thereby potentially associated with cell growth regulation, whereas metabolites/intermediates of TCA, oxidative phosphorylation (aspartate, glutamate, succinate, malate, fumarate and citrate), intracellular NAD+/NADH ratio, and glutathione metabolic pathways were observed to be upregulated in stationary-phase and hence potentially associated with increased cell-specific productivity in CHO bioprocess. Moreover, each of technique has its own bias towards metabolite identification, indicating their complementarity, along with a number of critical gaps in the CHO metabolomics pipeline and hence first time discussed here to identify their potential remedies. This knowledge may help in future study designs to improve the metabolomic coverage facilitating identification of the metabolites/pathways which might get missed otherwise and explore the full potential of metabolomics for improving the CHO bioprocess performances.

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Defects in Galactose Metabolism and Glycoconjugate Biosynthesis in a UDP-Glucose Pyrophosphorylase-Deficient Cell Line Are Reversed by Adding Galactose to the Growth Medium

Cited by 8 publications

References 59 publications

PPA1 Deficiency Causes a Deranged Galactose Metabolism Recognizable in Neonatal Screening

PPA1 Deficiency Causes a Deranged Galactose Metabolism Recognizable in Neonatal Screening

Poly‐pathways metabolomics for high‐yielding cordycepin of Cordyceps militaris

Advancements in CHO metabolomics: techniques, current state and evolving methodologies

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