Real-time monitoring of the sialic acid biosynthesis pathway by NMR

Gorenflos, Jacob; Schmieder, Peter; Kemnitz-Hassanin, Kristin; Asikoglu, Hatice Ceyda; Celik, Arif; Stieger, Christian E.; Fiedler, Dorothea; Hinderlich, Stephan; Hackenberger, Christian P. R.

doi:10.1039/d2sc06986e

Cited by 8 publications

(7 citation statements)

References 55 publications

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“…While a recent study implies, that exogenous ManNAc is phosphorylated by NAGK ( Gorenflos Lopez et al. 2023 ), our results show that even with Nagk being expressed in C2C12 Gne-KO cells, there is no ManNAc phosphorylation.…”

Section: Discussioncontrasting

confidence: 93%

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GNE deficiency impairs Myogenesis in C2C12 cells and cannot be rescued by ManNAc supplementation

Neu,

Weilepp,

Bork

et al. 2024

Glycobiology

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GNE myopathy (GNEM) is a late-onset muscle atrophy, caused by mutations in the gene for the key enzyme of sialic acid biosynthesis, UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE). With an incidence of one to nine cases per million it is an ultra-rare, so far untreatable, autosomal recessive disease. Several attempts have been made to treat GNEM patients by oral supplementation with sialic acid precursors (e.g. N-acetylmannosamine, ManNAc) to restore sarcolemmal sialylation and muscle strength. In most studies, however, no significant improvement was observed. The lack of a suitable mouse model makes it difficult to understand the exact pathomechanism of GNEM and many years of research have failed to identify the role of GNE in skeletal muscle due to the lack of appropriate tools. We established a CRISPR/Cas9-mediated Gne-knockout cell line using murine C2C12 cells to gain insight into the actual role of the GNE enzyme and sialylation in a muscular context. The main aspect of this study was to evaluate the therapeutic potential of ManNAc and N-acetylneuraminic acid (Neu5Ac). Treatment of Gne-deficient C2C12 cells with Neu5Ac, but not with ManNAc, showed a restoration of the sialylation level back to wild type levels–albeit only with long-term treatment, which could explain the rather low therapeutic potential. We furthermore highlight the importance of sialic acids on myogenesis, for C2C12 Gne-knockout myoblasts lack the ability to differentiate into mature myotubes.

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Section: Discussioncontrasting

confidence: 93%

“…It has been shown that the N-acetylglucosmaine kinase (NAGK) is able to phosphorylate ManNAc, and is thus believed to bypass the lack of GNE kinase activity in GNE-deficient cells ( Allen and Walker 1980 ). A recent study also suggests that external ManNAc is mainly phosphorylated by NAGK ( Gorenflos Lopez et al. 2023 ).…”

Section: Resultsmentioning

confidence: 93%

GNE deficiency impairs Myogenesis in C2C12 cells and cannot be rescued by ManNAc supplementation

Neu,

Weilepp,

Bork

et al. 2024

Glycobiology

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“…With wt-NAGK, pS76-NAGK, and ppS76-NAGK in hand, we next wanted to evaluate how the different phosphorylation modes influenced the enzymatic activity of NAGK. To do so, a standard kinase assay was set up using GlcNAc as a substrate and monitoring adenosine-5′-triphosphate (ATP) consumption (Figure a) . At low enzyme concentration (1 nM), unphosphorylated wt-NAGK displayed a robust activity of 260 nmol/min/ng.…”

Section: Resultsmentioning

confidence: 99%

“…To do so, a standard kinase assay was set up using GlcNAc as a substrate and monitoring adenosine-5′triphosphate (ATP) consumption (Figure 3a). 28 At low enzyme concentration (1 nM), unphosphorylated wt-NAGK displayed a robust activity of 260 nmol/min/ng. By contrast, pSer76-NAGK showed no activity at 1 nM enzyme concentration.…”

Section: Synthesis Of Pyrophosphorylated Nagkmentioning

confidence: 95%

An Uncommon Phosphorylation Mode Regulates the Activity and Protein Interactions of N-Acetylglucosamine Kinase

Celik,

Beyer,

Fiedler

2024

J. Am. Chem. Soc.

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While the function of protein phosphorylation in eukaryotic cell signaling is well established, the role of a closely related modification, protein pyrophosphorylation, is just starting to surface. A recent study has identified several targets of endogenous protein pyrophosphorylation in mammalian cell lines, including N-acetylglucosamine kinase (NAGK). Here, a detailed functional analysis of NAGK phosphorylation and pyrophosphorylation on serine 76 (S76) has been conducted. This analysis was enabled by using amber codon suppression to obtain phosphorylated pS76-NAGK, which was subsequently converted to site-specifically pyrophosphorylated NAGK (ppS76-NAGK) with a phosphorimidazolide reagent. A significant reduction in GlcNAc kinase activity was observed upon phosphorylation and near-complete inactivation upon pyrophosphorylation. The formation of ppS76-NAGK proceeded via an ATP-dependent autocatalytic process, and once formed, ppS76-NAGK displayed notable stability toward dephosphorylation in mammalian cell lysates. Proteomic examination unveiled a distinct set of protein− protein interactions for ppS76-NAGK, suggesting an alternative function, independent of its kinase activity. Overall, a significant regulatory role of pyrophosphorylation on NAGK activity was uncovered, providing a strong incentive to investigate the influence of this unusual phosphorylation mode on other kinases.

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“…The primary hits were validated with a plate reader‐independent and orthogonal NMR‐assay [18] . The N ‐acetyl methyl of UDP‐GlcNAc and ManNAc were found to display characteristic chemical shifts around 2.1–1.9 ppm (Supplementary Figure 12).…”

Section: Resultsmentioning

confidence: 99%

Small Molecules Targeting Human UDP‐GlcNAc 2‐Epimerase

Gorenflos López,

Dornan,

Boback

et al. 2023

ChemBioChem

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Uridine diphosphate N‐acetylglucosamine 2‐epimerase (GNE) is a key enzyme in the sialic acid biosynthesis pathway. Sialic acids are primarily terminal carbohydrates on glycans and play fundamental roles in health and disease. In search of effective GNE inhibitors not based on a carbohydrate scaffold, we performed a high‐throughput screening campaign of 68,640 drug‐like small molecules against recombinant GNE using a UDP detection assay. We validated nine of the primary actives with an orthogonal real‐time NMR assay and verified their IC50 values in the low micromolar to nanomolar range manually. Stability and solubility studies revealed three compounds for further evaluation. Thermal shift assays, analytical size exclusion and interferometric scattering microscopy demonstrated that the GNE inhibitors acted on the oligomeric state of the protein. Finally, hydrogen‐deuterium exchange mass spectrometry (HDX‐MS) revealed which sections of GNE were shifted upon the addition of the inhibitors. In summary, we have identified three small molecules as GNE inhibitors with high potency in vitro, which serve as promising candidates to modulate sialic acid biosynthesis in more complex systems

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Real-time monitoring of the sialic acid biosynthesis pathway by NMR

Abstract: Sialic acids are part of the outermost component of the glycocalyx of all vertebrates; as such, they are fundamental markers in physiological and pathological processes. In this study, we introduce...

Cited by 8 publications

References 55 publications

GNE deficiency impairs Myogenesis in C2C12 cells and cannot be rescued by ManNAc supplementation

GNE deficiency impairs Myogenesis in C2C12 cells and cannot be rescued by ManNAc supplementation

An Uncommon Phosphorylation Mode Regulates the Activity and Protein Interactions of N-Acetylglucosamine Kinase

Small Molecules Targeting Human UDP‐GlcNAc 2‐Epimerase

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