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Three forms of muscular dystrophy-dystroglycanopathies are linked to the ribitol pathway. These include mutations in the isoprenoid synthase domain-containing protein ( ISPD ), fukutin-related protein ( FKRP ), and fukutin ( FKTN ) genes. The aforementioned enzymes are required for generation of the ribitol phosphate linkage in the O-glycan of alpha-dystroglycan. Mild cases of dystroglycanopathy present with slowly progressive muscle weakness, while in severe cases the eyes and brain are also involved. Previous research showed that ribose increased the intracellular concentrations of cytidine diphosphate-ribitol (CDP-ribitol) and had a therapeutic effect. Here, we report the safety and effects of oral ribose supplementation during 6 months in a patient with limb girdle muscular dystrophy type 2I (LGMD2I) due to a homozygous FKRP mutation. Ribose was well tolerated in doses of 9 g or 18 g/day. Supplementation with 18 g of ribose resulted in a decrease of creatine kinase levels of 70%. Moreover, metabolomics showed a significant increase in CDP-ribitol levels with 18 g of ribose supplementation ( p  < 0.001). Although objective improvement in clinical and patient-reported outcome measures was not observed, the patient reported subjective improvement of muscle strength, fatigue, and pain. This case study indicates that ribose supplementation in patients with dystroglycanopathy is safe and highlights the importance for future studies regarding its potential effects., Competing Interests: The authors declare no conflicts of interest., (© 2024 The Authors. JIMD Reports published by John Wiley & Sons Ltd on behalf of SSIEM.)

Metabolism not only produces energy necessary for the cell but is also a key regulator of several cellular functions, including pluripotency and self-renewal. Nucleotide sugars (NSs) are activated sugars that link glucose metabolism with cellular functions via protein N-glycosylation and O-GlcNAcylation. Thus, understanding how different metabolic pathways converge in the synthesis of NSs is critical to explore new opportunities for metabolic interference and modulation of stem cell functions. Tracer-based metabolomics is suited for this challenge, however chemically-defined, customizable media for stem cell culture in which nutrients can be replaced with isotopically labeled analogs are scarcely available. Here, we established a customizable flux-conditioned E8 (FC-E8) medium that enables stem cell culture with stable isotopes for metabolic tracing, and a dedicated liquid chromatography mass-spectrometry (LC-MS/MS) method targeting metabolic pathways converging in NS biosynthesis. By 13 C 6 -glucose feeding, we successfully traced the time-course of carbon incorporation into NSs directly via glucose, and indirectly via other pathways, such as glycolysis and pentose phosphate pathways, in induced pluripotent stem cells (hiPSCs) and embryonic stem cells. Then, we applied these tools to investigate the NS biosynthesis in hiPSC lines from a patient affected by deficiency of phosphoglucomutase 1 (PGM1), an enzyme regulating the synthesis of the two most abundant NSs, UDP-glucose and UDP-galactose.

Phosphoglucomutase 1 (PGM1) is a key enzyme for the regulation of energy metabolism from glycogen and glycolysis, as it catalyzes the interconversion of glucose 1-phosphate and glucose 6-phosphate. PGM1 deficiency is an autosomal recessive disorder characterized by a highly heterogenous clinical spectrum, including hypoglycemia, cleft palate, liver dysfunction, growth delay, exercise intolerance, and dilated cardiomyopathy. Abnormal protein glycosylation has been observed in this disease. Oral supplementation with D-galactose efficiently restores protein glycosylation by replenishing the lacking pool of UDP-galactose, and rescues some symptoms, such as hypoglycemia, hepatopathy, and growth delay. However, D-galactose effects on skeletal muscle and heart symptoms remain unclear. In this study, we established an in vitro muscle model for PGM1 deficiency to investigate the role of PGM1 and the effect of D-galactose on nucleotide sugars and energy metabolism. Genome-editing of C2C12 myoblasts via CRISPR/Cas9 resulted in Pgm1 (mouse homologue of human PGM1 , according to updated nomenclature) knockout clones, which showed impaired maturation to myotubes. No difference was found for steady-state levels of nucleotide sugars, while dynamic flux analysis based on 13 C6-galactose suggested a block in the use of galactose for energy production in knockout myoblasts. Subsequent analyses revealed a lower basal respiration and mitochondrial ATP production capacity in the knockout myoblasts and myotubes, which were not restored by D-galactose. In conclusion, an in vitro mouse muscle cell model has been established to study the muscle-specific metabolic mechanisms in PGM1 deficiency, which suggested that galactose was unable to restore the reduced energy production capacity.

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., Competing Interests: HS, BN, BU, BA, IM, PM, MK, SB, KA, RV, DL, MT, HB, VP No competing interests declared, (© 2023, Scott, Novikov et al.)

The sugar fucose is expressed on mammalian cell membranes as part of glycoconjugates and mediates essential physiological processes. The aberrant expression of fucosylated glycans has been linked to pathologies such as cancer, inflammation, infection, and genetic disorders. Tools to modulate fucose expression on living cells are needed to elucidate the biological role of fucose sugars and the development of potential therapeutics. Herein, we report a class of fucosylation inhibitors directly targeting de novo GDP-fucose biosynthesis via competitive GMDS inhibition. We demonstrate that cell permeable fluorinated rhamnose 1-phosphate derivatives (Fucotrim I & II) are metabolic prodrugs that are metabolized to their respective GDP-mannose derivatives and efficiently inhibit cellular fucosylation., (© 2021. The Author(s).)

Fucosylation of glycans impacts a myriad of physiological and pathological processes. Inhibition of fucose expression emerges as a potential therapeutic avenue for example in cancer, inflammation, and infection. In this study, we found that protected 2-fluorofucose 1-phosphate efficiently inhibits cellular fucosylation with a four to seven times higher potency than known inhibitor 2FF, independently of the anomeric stereochemistry. Nucleotide sugar analysis revealed that both the α- and β-GDP-2FF anomers are formed inside the cell. In conclusion, we developed A2FF1P and B2FF1P as potent new tools for studying the role of fucosylation in health and disease and they are potential therapeutic candidates., (© 2020 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Nucleotide sugars (NS) are fundamental molecules in life and play a key role in glycosylation reactions and signal conduction. Several pathways are involved in the synthesis of NS. The Leloir pathway, the main pathway for galactose metabolism, is crucial for production of uridine diphosphate (UDP)-glucose and UDP-galactose. The most common metabolic disease affecting this pathway is galactose-1-phosphate uridylyltransferase (GALT) deficiency, that despite a lifelong galactose-restricted diet, often results in chronically debilitating complications. Alterations in the levels of UDP-sugars leading to galactosylation abnormalities have been hypothesized as a key pathogenic factor. However, UDP-sugar levels measured in patient cell lines have shown contradictory results. Other NS that might be affected, differences throughout development, as well as tissue specific profiles have not been investigated. Using recently established UHPLC-MS/MS technology, we studied the complete NS profiles in wildtype and galt knockout zebrafish (Danio rerio). Analyses of UDP-hexoses, UDP-hexosamines, CMP-sialic acids, GDP-fucose, UDP-glucuronic acid, UDP-xylose, CDP-ribitol, and ADP-ribose profiles at four developmental stages and in tissues (brain and gonads) in wildtype zebrafish revealed variation in NS levels throughout development and differences between examined tissues. More specifically, we found higher levels of CMP-N-acetylneuraminic acid, GDP-fucose, UDP-glucuronic acid, and UDP-xylose in brain and of CMP-N-glycolylneuraminic acid in gonads. Analysis of the same NS profiles in galt knockout zebrafish revealed no significant differences from wildtype. Our findings in galt knockout zebrafish, even when challenged with galactose, do not support a role for abnormalities in UDP-glucose or UDP-galactose as a key pathogenic factor in GALT deficiency, under the tested conditions., (© 2020 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)