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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. Aberrant expression of fucosylated glycans has been linked to several disease states. Control of fucose expression on live cells is needed to aid research and therapy development. Here the authors report on the development of a class of fucosylation metabolic prodrug inhibitors and demonstrated inhibition of cellular fucosylation.

Intestinal P-glycoprotein (P-gp/ ABCB1 ) is a key barrier limiting xenobiotic absorption, yet its functional decline with aging is poorly understood. Here, we show that gut microbiota dysbiosis contributes to age-associated P-gp deficiency. Integrated multi-omics analyses of human cohorts and murine models identify Odoribacter splanchnicus ( O. splanchnicus ) as a key commensal species whose depletion impairs intestinal P-gp function. Mechanistically, O. splanchnicus encodes GDP-mannose 4, 6-dehydratase (GMDS) and GDP-L-fucose synthase (TSTA3), enabling microbial biosynthesis of GDP-L-fucose. This metabolite directly promotes phosphorylation of the eukaryotic translation initiation factor 4E (eIF4E) and activates c-Jun-driven ABCB1 expression, thereby restoring xenobiotic efflux. These findings establish a microbiota-metabolite-transporter signaling axis that maintains intestinal detoxification, suggesting that targeting either microbes or metabolites could help prevent adverse drug reactions in older adults. Here, with multi-omics analyses of human cohorts and murine models, the authors identify Odoribacter splanchnicus depletion to impair intestinal P-glycoprotein function via loss of microbial, GDP-L-fucose, which in turn promotes eIF4E phosphorylation and c-Jun–driven ABCB1 expression, maintaining xenobiotic efflux.

Notch is a cell-surface receptor that controls cell-fate decisions and is regulated by O-glycans attached to epidermal growth factor-like (EGF) repeats in its extracellular domain. Protein O-fucosyltransferase 1 (Pofut1) modifies EGF repeats with O-fucose and is essential for Notch signaling. Constitutive activation of Notch signaling has been associated with a variety of human malignancies. Therefore, tools that inhibit Notch activity are being developed as cancer therapeutics. To this end, we screened L-fucose analogs for their effects on Notch signaling. Two analogs, 6-alkynyl and 6-alkenyl fucose, were substrates of Pofut1 and were incorporated directly into Notch EGF repeats in cells. Both analogs were potent inhibitors of binding to and activation of Notch1 by Notch ligands Dll1 and Dll4, but not by Jag1. Mutagenesis and modeling studies suggest that incorporation of the analogs into EGF8 of Notch1 markedly reduces the ability of Delta ligands to bind and activate Notch1.

GDP-fucose is synthesised via two pathways: de novo and salvage. The first uses GDP-mannose as a substrate, and the second uses free fucose. To date, these pathways have been considered to work separately and not to have an influence on each other. We report the mutual response of the de novo and salvage pathways to the lack of enzymes from a particular route of GDP-fucose synthesis. We detected different efficiencies of GDP-fucose and fucosylated structure synthesis after a single inactivation of enzymes of the de novo pathway. Our study demonstrated the unequal influence of the salvage enzymes on the production of GDP-fucose by enzymes of the de novo biosynthesis pathway. Simultaneously, we detected an elevated level of one of the enzymes of the de novo pathway in the cell line lacking the enzyme of the salvage biosynthesis pathway. Additionally, we identified dissimilarities in fucose uptake between cells lacking TSTA3 and GMDS proteins.

Lewis X (Lex)-containing glycans play important roles in numerous cellular processes. However, the absence of robust, facile, and cost-effective methods for the synthesis of Lex and its structurally related analogs has severely hampered the elucidation of the specific functions of these glycan epitopes. Here we demonstrate that chemically defined guanidine 5'-diphosphate-β-L-fucose (GDP-fucose), the universal fucosyl donor, the Lex trisaccharide, and their C-5 substituted derivatives can be synthesized on preparative scales, using a chemoenzymatic approach. This method exploits L-fucokinase/GDP-fucose pyrophosphorylase (FKP), a bifunctional enzyme isolated from Bacteroides fragilis 9343, which converts L-fucose into GDP-fucose via a fucose-1-phosphate (Fuc-1-P) intermediate. Combining the activities of FKP and a Helicobacter pylori α1,3 fucosyltransferase, we prepared a library of Lex trisaccharide glycans bearing a wide variety of functional groups at the fucose C-5 position. These neoglycoconjugates will be invaluable tools for studying Lex-mediated biological processes.

The objectives of this study were to employ response surface methodology (RSM) to investigate and optimize the effect of ultrasound-assisted extraction (UAE) variables, temperature, time and amplitude on the yields of polysaccharides (fucose and total glucans) and antioxidant activities (ferric reducing antioxidant power (FRAP) and 1,1-diphenyl-2-picryl-hydrazyl radical scavenging activity (DPPH)) from Laminaria digitata, and to explore the suitability of applying the optimum UAE conditions for L. digitata to other brown macroalgae (L. hyperborea and Ascophyllum nodosum). The RSM with three-factor, four-level Box-Behnken Design (BBD) was used to study and optimize the extraction variables. A second order polynomial model fitted well to the experimental data with R2 values of 0.79, 0.66, 0.64, 0.73 for fucose, total glucans, FRAP and DPPH, respectively. The UAE parameters studied had a significant influence on the levels of fucose, FRAP and DPPH. The optimised UAE conditions (temperature = 76 °C, time = 10 min and amplitude = 100%) achieved yields of fucose (1060.7 ± 70.6 mg/100 g dried seaweed (ds)), total glucans (968.6 ± 13.3 mg/100 g ds), FRAP (8.7 ± 0.5 µM trolox/mg freeze-dried extract (fde)) and DPPH (11.0 ± 0.2%) in L. digitata. Polysaccharide rich extracts were also attained from L. hyperborea and A. nodosum with variable results when utilizing the optimum UAE conditions for L. digitata.

The key role played by fucose in glycoprotein and cellular function has prompted significant research toward identifying recombinant and biochemical strategies for blocking its incorporation into proteins and membrane structures. Technologies surrounding engineered cell lines have evolved for the inhibition of in vitro fucosylation, but they are not applicable for in vivo use and drug development. To address this, we screened a panel of fucose analogues and identified 2-fluorofucose and 5-alkynylfucose derivatives that depleted cells of GDP-fucose, the substrate used by fucosyltransferases to incorporate fucose into protein and cellular glycans. The inhibitors were used in vitro to generate fucose-deficient antibodies with enhanced antibody-dependent cellular cytotoxicity activities. When given orally to mice, 2-fluorofucose inhibited fucosylation of endogenously produced antibodies, tumor xenograft membranes, and neutrophil adhesion glycans. We show that oral 2-fluorofucose treatment afforded complete protection from tumor engraftment in a syngeneic tumor vaccine model, inhibited neutrophil extravasation, and delayed the outgrowth of tumor xenografts in immune-deficient mice. The results point to several potential therapeutic applications for molecules that selectively block the endogenous generation of fucosylated glycan structures.

Plants mount coordinated immune responses to defend themselves against pathogens. However, the cellular components required for plant immunity are not fully understood. The jasmonate-mimicking coronatine (COR) toxin produced by Pseudomonas syringae pv. tomato (Pst) DC3000 functions to overcome plant immunity. We previously isolated eight Arabidopsis (scord) mutants that exhibit increased susceptibility to a COR-deficient mutant of Pst DC3000. Among them, the scord6 mutant exhibits defects both in stomatal closure response and in restricting bacterial multiplication inside the apoplast. However, the identity of SCORD6 remained elusive. In this study, we aim to identify the SCORD6 gene. We identified SCORD6 via next-generation sequencing and found it to be MURUS1 (MUR1), which is involved in the biosynthesis of GDP-L-fucose. Discovery of SCORD6 as MUR1 led to a series of experiments that revealed a multi-faceted role of L-fucose biosynthesis in stomatal and apoplastic defenses as well as in patterntriggered immunity and effector-triggered immunity, including glycosylation of patternrecognition receptors. Furthermore, compromised stomatal and/or apoplastic defenses were observed in mutants of several fucosyltransferases with specific substrates (e.g. O-glycan, N-glycan or the DELLA transcriptional repressors). Collectively, these results uncover a novel and broad role of L-fucose and protein fucosylation in plant immunity.

Congenital disorders of glycosylation are a genetically and phenotypically heterogeneous family of diseases affecting the co‐ and posttranslational modification of proteins. Using exome sequencing, we detected biallelic variants in GFUS (NM_003313.4) c.[632G>A];[659C>T] (p.[Gly211Glu];[Ser220Leu]) in a patient presenting with global developmental delay, mild coarse facial features and faltering growth. GFUS encodes GDP‐L‐fucose synthase, the terminal enzyme in de novo synthesis of GDP‐L‐fucose, required for fucosylation of N‐ and O‐glycans. We found reduced GFUS protein and decreased GDP‐L‐fucose levels leading to a general hypofucosylation determined in patient's glycoproteins in serum, leukocytes, thrombocytes and fibroblasts. Complementation of patient fibroblasts with wild‐type GFUS cDNA restored fucosylation. Making use of the GDP‐L‐fucose salvage pathway, oral fucose supplementation normalized fucosylation of proteins within 4 weeks as measured in serum and leukocytes. During the follow‐up of 19 months, a moderate improvement of growth was seen, as well as a clear improvement of cognitive skills as measured by the Kaufmann ABC and the Nijmegen Pediatric CDG Rating Scale. In conclusion, GFUS‐CDG is a new glycosylation disorder for which oral L‐fucose supplementation is promising. Synopsis A novel type of congenital disorder of glycosylation (CDG) was identified in a child carrying biallelic variants in GDP‐L‐fucose synthase (GFUS). Oral L‐fucose supplementation resulted in clinical improvements. The index patient presented with stunted growth and no speech at the age of 3 6/12 years, coarse facial features, aversion to feeding, and recurrent vomiting on tube feeding. Biallelic variants in GFUS lead to a reduced level of GDP‐L‐fucose and subsequently to a general hypofucosylation of proteins. GFUS deficiency can be bypassed by oral L‐fucose supplementation triggering the salvage pathway for the generation of GDP‐L‐fucose. After the fucose therapy, the growth parameters have stabilized, and the patient started to speak and showed improved attention span and cognitive skills. Graphical Abstract A novel type of congenital disorder of glycosylation (CDG) was identified in a child carrying biallelic variants in GDP‐L‐fucose synthase (GFUS). Oral L‐fucose supplementation resulted in clinical improvements.

Background Core fucosylation (addition of fucose in α-1,6-linkage to core N-acetylglucosamine of N-glycans) catalyzed by fucosyltransferase 8 (FUT8) is critical for signaling receptors involved in many physiological and pathological processes such as cell growth, adhesion, and tumor metastasis. Transforming growth factor-β (TGF-β)-induced epithelial–mesenchymal transition (EMT) regulates the invasion and metastasis of breast tumors. However, whether receptor core fucosylation affects TGF-β signaling during breast cancer progression remains largely unknown. Method In this study, gene expression profiling and western blot were used to validate the EMT-associated expression of FUT8. Lentivirus-mediated gain-of-function study, short hairpin RNA (shRNA) or CRISPR/Cas9-mediated loss-of-function studies and pharmacological inhibition of FUT8 were used to elucidate the molecular function of FUT8 during TGF-β-induced EMT in breast carcinoma cells. In addition, lectin blot, luciferase assay, and in vitro ligand binding assay were employed to demonstrate the involvement of FUT8 in the TGF-β1 signaling pathway. The role of FUT8 in breast cancer migration, invasion, and metastasis was confirmed using an in vitro transwell assay and mammary fat pad xenograft in vivo tumor model. Results Gene expression profiling analysis revealed that FUT8 is upregulated in TGF-β-induced EMT; the process was associated with the migratory and invasive abilities of several breast carcinoma cell lines. Gain-of-function and loss-of-function studies demonstrated that FUT8 overexpression stimulated the EMT process, whereas FUT8 knockdown suppressed the invasiveness of highly aggressive breast carcinoma cells. Furthermore, TGF-β receptor complexes might be core fucosylated by FUT8 to facilitate TGF-β binding and enhance downstream signaling. Importantly, FUT8 inhibition suppressed the invasive ability of highly metastatic breast cancer cells and impaired their lung metastasis. Conclusions Our results reveal a positive feedback mechanism of FUT8-mediated receptor core fucosylation that promotes TGF-β signaling and EMT, thus stimulating breast cancer cell invasion and metastasis.