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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.

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.

Fucosylation is an oligosaccharide modification that plays an important role in immune response and malignancy, and specific fucosyltransferases (FUTs) catalyze the three types of fucosylations: core-type, Lewis type, and H type. FUTs regulate cancer proliferation, invasiveness, and resistance to chemotherapy by modifying the glycosylation of signaling receptors. Oligosaccharides on PD-1/PD-L1 proteins are specifically fucosylated, leading to functional modifications. Expression of FUTs is upregulated in renal cell carcinoma, bladder cancer, and prostate cancer. Aberrant fucosylation in prostate-specific antigen (PSA) could be used as a novel biomarker for prostate cancer. Furthermore, elucidation of the biological function of fucosylation could result in the development of novel therapeutic targets. Further studies are needed in the field of fucosylation glycobiology in urological malignancies.

Fucosyllactoses (FL), including 2′‐fucosyllactose (2′‐FL) and 3‐fucosyllactose (3‐FL), have garnered considerable interest for their value in newborn formula and pharmaceuticals. In this study, an engineered Escherichia coli was developed for high‐titer FL biosynthesis by introducing multi‐level metabolic engineering strategies, including (1) individual construction of the 2′/3‐FL‐producing strains through gene combination optimization of the GDP‐L‐fucose module; (2) screening of rate‐limiting enzymes (α‐1,2‐fucosyltransferase and α‐1,3‐fucosyltransferase); (3) analysis of critical intermediates and inactivation of competing pathways to redirect carbon fluxes to FL biosynthesis; (4) enhancement of the catalytic performance of rate‐limiting enzymes by the RBS screening, fusion peptides and multi‐copy gene cloning. The final strains EC49 and EM47 produced 9.36 g/L for 2′‐FL and 6.28 g/L for 3‐FL in shake flasks with a modified‐M9CA medium. Fed‐batch cultivations of the two strains generated 64.62 g/L of 2′‐FL and 40.68 g/L of 3‐FL in the 3‐L bioreactors, with yields of 0.65 mol 2′‐FL/mol lactose and 0.67 mol 3‐FL/mol lactose, respectively. This research provides a viable platform for other high‐value‐added compounds production in microbial cell factories. An engineered Escherichia coli was developed for high‐titer FL biosynthesis by introducing multi‐level metabolic engineering strategies. Combined with the optimization of metabolic pathways and the performance improvement of rate‐limiting enzymes, 64.62 g/L of 2 '‐FL and 40.68 g/L of 3‐FL were finally obtained in the 3‐L bioreactors.

Aberrant glycosylation is recognized as a cancer hallmark that is associated with cancer development and progression. In this study, the clinical relevance and significance of terminal fucose (TFG), by fucosyltransferase-1 (FUT1) in carcinogenesis and progression of cholangiocarcinoma (CCA) were demonstrated. TFG expression in human and hamster CCA tissues were determined using Ulex europaeus agglutinin-I (UEA-I) histochemistry. Normal bile ducts rarely expressed TFG while 47% of CCA human tissues had high TFG expression and was correlated with shorter survival of patients. In the CCA-hamster model, TFG was elevated in hyperproliferative bile ducts and gradually increased until CCA was developed. This evidence indicates the involvement of TFG in carcinogenesis and progression of CCA. The mechanistic insight was performed in 2 CCA cell lines. Suppression of TFG expression using siFUT1 or neutralizing the surface TFG with UEA-I significantly reduced migration, invasion and adhesion of CCA cells in correlation with the reduction of Akt/Erk signaling and epithelial-mesenchymal transition. A short pulse of EGF could stimulate Akt/Erk signaling via activation of EGF-EGFR cascade, however, decreasing TFG using siFUT1 or UEA-I treatment reduced the EGF-EGFR activation and Akt/Erk signaling. This evidence provides important insight into the relevant role and molecular mechanism of TFG in progression of CCA.

Fucosylation plays a fundamental role in maintaining cellular functions and biological processes across all animals. As a form of glycosylation, it involves the biochemical addition of fucose, a six-carbon monosaccharide, to biological molecules like lipids, proteins, and glycan chains. This modification is essential for optimizing cellular interactions required for receptor-ligand binding, cell adhesion, immune responses, and development. Disruptions in cellular fucose synthesis or in the mechanisms enabling its transfer to other molecules have been linked to human disease. Inherited defects in the fucosylation pathway are rare, with about thirty patients described. Through genome-wide association studies (GWAS), variants in fucosylation pathway genes have been associated with complex diseases such as glaucoma and stroke, and somatic mutations are often found in cancers. Recent studies have applied targeted genetic animal models to elucidate the mechanisms through which disruptions in fucosylation contribute to disease pathogenesis and progression. Key focus areas include GDP-fucose synthesis through de novo or salvage pathways, GDP-fucose transport into the Golgi and endoplasmic reticulum (ER), and its transfer by fucosyltransferases (FUTs) or protein O-fucosyltransferases (POFUTs) onto acceptor molecules. Loss or gain of function fucosylation gene mutations in animal models such as mice, zebrafish, and invertebrates have provided insights into some fucosylation disease pathogenesis. This review aims to bring together these findings, summarizing key insights from existing animal studies to possibly infer fucosylation disease mechanisms in humans.

Protein O ‐fucosylation is a post‐translational modification found on serine/threonine residues of thrombospondin type 1 repeats (TSR). The fucose transfer is catalysed by the enzyme protein O ‐fucosyltransferase 2 (POFUT2) and >40 human proteins contain the TSR consensus sequence for POFUT2‐dependent fucosylation. To better understand O ‐fucosylation on TSR, we carried out a structural and functional analysis of human POFUT2 and its TSR substrate. Crystal structures of POFUT2 reveal a variation of the classical GT‐B fold and identify sugar donor and TSR acceptor binding sites. Structural findings are correlated with steady‐state kinetic measurements of wild‐type and mutant POFUT2 and TSR and give insight into the catalytic mechanism and substrate specificity. By using an artificial mini‐TSR substrate, we show that specificity is not primarily encoded in the TSR protein sequence but rather in the unusual 3D structure of a small part of the TSR. Our findings uncover that recognition of distinct conserved 3D fold motifs can be used as a mechanism to achieve substrate specificity by enzymes modifying completely folded proteins of very wide sequence diversity and biological function. This study presents the first crystal structure of human O ‐fucosyltransferase 2 bound to its substrate, the thrombospondin type I repeat present in numerous proteins. Functional analysis provides insight into the catalytic mechanism generating this post‐translational modification and structure encoded substrate specificity.

Background In endothelial cells, phospholipase C (PLC) β1-activated Ca 2+ is a crucial second messenger for the signaling pathways governing angiogenesis. PLCβ1 is inactivated by complexing with an intracellular protein called translin-associated factor X (TRAX). This study demonstrates specific interactions between Globo H ceramide (GHCer) and TRAX, which highlight a new angiogenic control through PLCβ1 activation. Methods Globo-series glycosphingolipids (GSLs), including GHCer and stage-specific embryonic antigen-3 ceramide (SSEA3Cer), were analyzed using enzyme-linked immunosorbent assay (ELISA) and Biacore for their binding with TRAX. Angiogenic activities of GSLs in human umbilical vein endothelial cells (HUVECs) were evaluated. Molecular dynamics (MD) simulation was used to study conformations of GSLs and their molecular interactions with TRAX. Fluorescence resonance energy transfer (FRET) analysis of HUVECs by confocal microscopy was used to validate the release of PLCβ1 from TRAX. Furthermore, the in vivo angiogenic activity of extracellular vesicles (EVs) containing GHCer was confirmed using subcutaneous Matrigel plug assay in mice. Results The results of ELISA and Biacore analysis showed a stable complex between recombinant TRAX and synthetic GHCer with K d of 40.9 nM. In contrast, SSEA3Cer lacking a fucose residue of GHCer at the terminal showed ~ 1000-fold decrease in the binding affinity. These results were consistent with their angiogenic activities in HUVECs. The MD simulation indicated that TRAX interacted with the glycan moiety of GHCer at amino acid Q223, Q219, L142, S141, and E216. At equilibrium the stable complex maintained 4.6 ± 1.3 H-bonds. TRAX containing double mutations with Q223A and Q219A lost its ability to interact with GHCer in both MD simulation and Biacore assays. Removal of the terminal fucose from GHCer to become SSEA3Cer resulted in decreased H-bonding to 1.2 ± 1.0 by the MD simulation. Such specific H-bonding was due to the conformational alteration in the whole glycan which was affected by the presence or absence of the fucose moiety. In addition, ELISA, Biacore, and in-cell FRET assays confirmed the competition between GHCer and PLCβ1 for binding to TRAX. Furthermore, the Matrigel plug assay showed robust vessel formation in the plug containing tumor-secreted EVs or synthetic GHCer, but not in the plug with SSEA3Cer. The FRET analysis also indicated the disruption of colocalization of TRAX and PLCβ1 in cells by GHCer derived from EVs. Conclusions Overall, the fucose residue in GHCer dictated the glycan conformation for its complexing with TRAX to release TRAX-sequestered PLCβ1, leading to Ca 2+ mobilization in endothelial cells and enhancing angiogenesis in tumor microenvironments.

The CUP-SHAPED COTYLEDON (CUC) transcription factors control plant boundary formation, thus allowing the emergence of novel growth axes. While the developmental roles of the CUC genes in different organs and across species are well characterized, upstream and downstream events that contribute to their function are still poorly understood. To identify new players in this network, we performed a suppressor screen of CUC2g-m4, a line overexpressing CUC2 that has highly serrated leaves. We identified a mutation that simplifies leaf shape and affects MURUS1 (MUR1), which is responsible for GDP-L-fucose production. Using detailed morphometric analysis, we show that GDP-L-fucose has an essential role in leaf shape acquisition by sustaining differential growth at the leaf margins. Accordingly, reduced CUC2 expression levels are observed in mur1 leaves. Furthermore, genetic analyses reveal a conserved role for GDPL- fucose in different developmental contexts where it contributes to organ separation in the same pathway as CUC2. Taken together, our results reveal that GDP-L-fucose is necessary for proper establishment of boundary domains in various developmental contexts.

Antibody-dependent cellular cytotoxicity (ADCC) is an important effector function determining the clinical efficacy of therapeutic antibodies. Core fucose removal from N-glycans on the Fc portion of immunoglobulin G (IgG) improves the binding affinity for Fcγ receptor IIIa (FcγRIIIa) and dramatically enhances ADCC. Our previous structural analyses revealed that Tyr-296 of IgG1-Fc plays a critical role in the interaction with FcγRIIIa, particularly in the enhanced FcγRIIIa binding of nonfucosylated IgG1. However, the importance of the Tyr-296 residue in the antibody in the interaction with various Fcγ receptors has not yet been elucidated. To further clarify the biological importance of this residue, we established comprehensive Tyr-296 mutants as fucosylated and nonfucosylated anti-CD20 IgG1s rituximab variants and examined their binding to recombinant soluble human Fcγ receptors: shFcγRI, shFcγRIIa, shFcγRIIIa, and shFcγRIIIb. Some of the mutations affected the binding of antibody to not only shFcγRIIIa but also shFcγRIIa and shFcγRIIIb, suggesting that the Tyr-296 residue in the antibody was also involved in interactions with FcγRIIa and FcγRIIIb. For FcγRIIIa binding, almost all Tyr-296 variants showed lower binding affinities than the wild-type antibody, irrespective of their core fucosylation, particularly in Y296K and Y296P. Notably, only the Y296W mutant showed improved binding to FcγRIIIa. The 3.00 Å-resolution crystal structure of the nonfucosylated Y296W mutant in complex with shFcγRIIIa harboring two N-glycans revealed that the Tyr-to-Trp substitution increased the number of potential contact atoms in the complex, thus improving the binding of the antibody to shFcγRIIIa. The nonfucosylated Y296W mutant retained high ADCC activity, relative to the nonfucosylated wild-type IgG1, and showed greater binding affinity for FcγRIIa. Our data may improve our understanding of the biological importance of human IgG1-Fc Tyr-296 in interactions with various Fcγ receptors, and have applications in the modulation of the IgG1-Fc function of therapeutic antibodies.