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Core fucosylation is catalyzed by α‐1,6‐fucosyltransferase (FUT8), which fucosylates the innermost GlcNAc of N‐glycans. Given the association of FUT8 with various diseases, including cancer, selective FUT8 inhibitors applicable to in vivo or cell‐based systems are highly sought‐after. Herein, we report the discovery of a compound that selectively inhibits FUT8 in cell‐based assays. High‐throughput screening revealed a FUT8‐inhibiting pharmacophore, and further structural optimization yielded an inhibitor with a KD value of 49 nM. Notably, this binding occurs only in the presence of GDP (a product of the enzymatic reaction catalyzed by FUT8). Mechanistic studies suggested that this inhibitor generates a highly reactive naphthoquinone methide derivative at the binding site in FUT8, which subsequently reacts with FUT8. Furthermore, prodrug derivatization of this inhibitor improved its stability, enabling suppression of core fucose expression and subsequent EGFR and T‐cell signaling in cell‐based assays, paving the way for the development of drugs targeting core fucosylation. [ABSTRACT FROM AUTHOR]

Summary: Microalgae are considered as attractive expression systems for the production of biologics. As photosynthetic unicellular organisms, they do not require costly and complex media for growing and are able to secrete proteins and perform protein glycosylation. Some biologics have been successfully produced in the green microalgae Chlamydomonas reinhardtii. However, post‐translational modifications like glycosylation of these Chlamydomonas‐made biologics have poorly been investigated so far. Therefore, in this study, we report on the first structural investigation of glycans linked to human erythropoietin (hEPO) expressed in a wild‐type C. reinhardtii strain and mutants impaired in key Golgi glycosyltransferases. The glycoproteomic analysis of recombinant hEPO (rhEPO) expressed in the wild‐type strain demonstrated that the three N‐glycosylation sites are 100% glycosylated with mature N‐glycans containing four to five mannose residues and carrying core xylose, core fucose and O‐methyl groups. Moreover, expression in C. reinhardtii insertional mutants defective in xylosyltransferases A and B and fucosyltransferase resulted in drastic decreases of core xylosylation and core fucosylation of glycans N‐linked to the rhEPOs, thus demonstrating that this strategy offers perspectives for humanizing the N‐glycosylation of the Chlamydomonas‐made biologics. [ABSTRACT FROM AUTHOR]

Down syndrome (DS) is characterized by lowered immune competence and premature aging. We previously showed decreased antibody response following SARS‐CoV‐2 vaccination in adults with DS. IgG1 Fc glycosylation patterns are known to affect the effector function of IgG and are associated with aging. Here, we compare total and anti‐spike (S) IgG1 glycosylation patterns following SARS‐CoV‐2 vaccination in DS and healthy controls (HC). Total and anti‐Spike IgG1 Fc N‐glycan glycoprofiles were measured in non‐exposed adults with DS and controls before and after SARS‐CoV‐2 vaccination by liquid chromatography–mass spectrometry (LC–MS) of Fc glycopeptides. We recruited N = 44 patients and N = 40 controls. We confirmed IgG glycosylation patterns associated with aging in HC and showed premature aging in DS. In DS, we found decreased galactosylation (50.2% vs. 59.0%) and sialylation (6.7% vs. 8.5%) as well as increased fucosylation (97.0% vs. 94.6%) of total IgG. Both cohorts showed similar bisecting GlcNAc of total and anti‐S IgG1 with age. In contrast, anti‐S IgG1 of DS and HC showed highly comparable glycosylation profiles 28 days post vaccination. The IgG1 glycoprofile in DS exhibits strong premature aging. The combination of an early decrease in IgG1 Fc galactosylation and sialylation and increase in fucosylation is predicted to reduce complement activity and decrease FcγRIII binding and subsequent activation, respectively. The altered glycosylation patterns, combined with decreased antibody concentrations, help us understand the susceptibility to severe infections in DS. The effect of premature aging highlights the need for individuals with DS to receive tailored vaccines and/or vaccination schedules. [ABSTRACT FROM AUTHOR]

Cell engineering strategies typically rely on energy‐consuming overexpression of genes or radical gene‐knock out. Both strategies are not particularly convenient for the generation of slightly modulated phenotypes, as needed in biosimilar development of for example differentially fucosylated monoclonal antibodies (mAbs). Recently, transiently transfected small noncoding microRNAs (miRNAs), known to be regulators of entire gene networks, have emerged as potent fucosylation modulators in Chinese hamster ovary (CHO) production cells. Here, we demonstrate the applicability of stable miRNA overexpression in CHO production cells to adjust the fucosylation pattern of mAbs as a model phenotype. For this purpose, we applied a miRNA chaining strategy to achieve adjustability of fucosylation in stable cell pools. In addition, we were able to implement recently developed artificial miRNAs (amiRNAs) based on native miRNA sequences into a stable CHO expression system to even further fine‐tune fucosylation regulation. Our results demonstrate the potential of miRNAs as a versatile tool to control mAb fucosylation in CHO production cells without adverse side effects on important process parameters. [ABSTRACT FROM AUTHOR]

Fucosyltransferases (Fut) regulate the fucosylation process associated with tumorogenesis in different cancer types. Ascitic fluid (AF) from patients diagnosed with advanced stage of epithelial ovarian cancer (EOC) is considered as a dynamic tumor microenvironment associated with poor prognosis. Previous studies from our laboratory showed increased fucosylation in SKOV‐3 and OVCAR‐3, cancer‐derived cell lines, when these cells were incubated with AFs derived from patients diagnosed with EOC. In the present work we studied three fucosyltransferases (Fut 2, Fut 4, and Fut 8) in SKOV‐3, OVCAR‐3 and CAOV‐3 cell lines in combination with five different AFs from patients diagnosed with this disease, confirming that all tested AFs increased fucosylation. Then, we demonstrate that mRNAs of these three enzymes were overexpressed in the three cell lines under treatment with AFs. SKOV‐3 showed the higher overexpression of Fut 2, Fut 4, and Fut 8 in comparison with the control condition. We further confirmed, in the SKOV‐3 cell line, by endpoint PCR, WB, and confocal microscopy, that the three enzymes were overexpressed, being Fut 4 the most overexpressed enzyme compared to Fut 2 and Fut 8. These enzymes were concentrated in vesicular structures with a homogeneous distribution pattern throughout the cytoplasm. Moreover, we found that among the three enzymes, only Fut 4 was located inside the nuclei. The nuclear location of Fut 4 was confirmed for the three cell lines. These results allow to propose Fut 2, Fut 4, and Fut 8 as potential targets for EOC treatment or as diagnostic tools for this disease. [ABSTRACT FROM AUTHOR]

Fucosylation is an important quality attribute for therapeutic antibodies. Afucosylated antibodies exhibit higher therapeutic efficacies than their fucosylated counterparts through antibody‐dependent cellular cytotoxicity (ADCC) mechanism. Since higher potency is beneficial in reducing dose or duration of the treatment, afucosylated antibodies have attracted a great deal of interest in biotherapeutics development. In this study, novel small molecules GDP‐D‐Rhamnose and its derivatives (Ac‐GDP‐D‐Rhamnose and rhamnose sodium phosphate) were synthesized to inhibit the enzyme in the GDP‐fucose synthesis pathway. Addition of these compounds into cell culture increased antibody afucosylation levels in a dose‐dependent manner and had no significant impact on other protein quality attributes. A novel and effective mechanism to generate afucosylated antibody is demonstrated for biologics discovery, analytical method development, process development, and other applications. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies (MAbs) are powerful therapeutic tools in modern medicine and represent a rapidly expanding multibillion USD market. While bioprocesses are generally well understood and optimized for MAbs, online quality control remains challenging. Notably, N‐glycosylation is a critical quality attribute of MAbs as it affects binding to Fcγ receptors (FcγRs), impacting the efficacy and safety of MAbs. Traditional N‐glycosylation characterization methods are ill‐suited for online monitoring of a bioreactor; in contrast, surface plasmon resonance (SPR) represents a promising avenue, as SPR biosensors can record MAb–FcγR interactions in real‐time and without labeling. In this study, we produced five lots of differentially glycosylated Trastuzumab (TZM) and finely characterized their glycosylation profile by HILIC‐UPLC chromatography. We then compared the interaction kinetics of these MAb lots with four FcγRs including FcγRIIA and FcγRIIB at 5°C and 25°C. When interacting with FcγRIIA/B at low temperature, the differentially glycosylated MAb lots exhibited distinct kinetic behaviors, contrary to room‐temperature experiments. Galactosylated TZM (1) and core fucosylated TZM (2) could be discriminated and even quantified using an analytical technique based on the area under the curve of the signal recorded during the dissociation phase of a SPR sensorgram describing the interaction with FcγRIIA (1) or FcγRII2B (2). Because of the rapidity of the proposed method (<5 min per measurement) and the small sample concentration it requires (as low as 30 nM, exact concentration not required), it could be a valuable process analytical technology for MAb glycosylation monitoring. [ABSTRACT FROM AUTHOR]

Fucosylation plays a critical role in cell‐to‐cell interactions and disease progression. However, the effects of fucosylation on splenocytes and their interactions with T cells remain unclear. In this study, we aimed to explore the transcriptome profiles of splenocytes deficient in fucosyltransferase (FUT) 1, an enzyme that mediates fucosylation, and investigate their impact on the proliferation and differentiation of T cells. We analysed and compared the transcriptomes of splenocytes isolated from Fut1 knockout (KO) mice and those from wild‐type (WT) mice using RNA‐seq. Additionally, we examined the effects of Fut1 KO splenocytes on CD4 T cell proliferation and differentiation, in comparison to WT splenocytes, and elucidated the mechanisms involved. The comparative analysis of transcriptomes between Fut1 KO and WT splenocytes revealed that thrombospondin‐1, among the genes related to immune response and inflammation, was the most highly downregulated gene in Fut1 KO splenocytes. The reduced expression of thrombospondin‐1 was further confirmed using qRT‐PCR and flow cytometry. In coculture experiments, Fut1 KO splenocytes promoted the proliferation of CD4 T cells and drove their differentiation toward Th1 and Th17 cells, compared with WT splenocytes. Moreover, the levels of IL‐2, IFN‐γ and IL‐17 were increased, while IL‐10 was decreased, in T cells cocultured with Fut1 KO splenocytes compared with those with WT splenocytes. These effects of Fut1 KO splenocytes on T cells were reversed when thrombospondin‐1 was replenished. Taken together, our results demonstrate that splenocytes with Fut1 deficiency promote CD4 T cell proliferation and Th1/Th17 differentiation at least in part through thrombospondin‐1 downregulation. [ABSTRACT FROM AUTHOR]

Summary: Land plants evolved multiple adaptations to restrict transpiration. However, the underlying molecular mechanisms are not sufficiently understood.We used an ozone‐sensitivity forward genetics approach to identify Arabidopsis thaliana mutants impaired in gas exchange regulation.High water loss from detached leaves and impaired decrease of leaf conductance in response to multiple stomata‐closing stimuli were identified in a mutant of MURUS1 (MUR1), an enzyme required for GDP‐l‐fucose biosynthesis. High water loss observed in mur1 was independent from stomatal movements and instead could be linked to metabolic defects. Plants defective in import of GDP‐l‐Fuc into the Golgi apparatus phenocopied the high water loss of mur1 mutants, linking this phenotype to Golgi‐localized fucosylation events. However, impaired fucosylation of xyloglucan, N‐linked glycans, and arabinogalactan proteins did not explain the aberrant water loss of mur1 mutants.Partial reversion of mur1 water loss phenotype by borate supplementation and high water loss observed in boron uptake mutants link mur1 gas exchange phenotypes to pleiotropic consequences of l‐fucose and boron deficiency, which in turn affect mechanical and morphological properties of stomatal complexes and whole‐plant physiology. Our work emphasizes the impact of fucose metabolism and boron uptake on plant–water relations. [ABSTRACT FROM AUTHOR]

Background: Congenital disorders of glycosylation (CDG) are a group of heterogeneous disorders caused by abnormal lipid or protein glycosylation. Variants in the FCSK gene have been reported to cause CDG. Defective FCSK‐induced CDG (FCSK–CDG) has only been reported previously in three unrelated children. Methods: In this study, we genetically and clinically examined a 3‐year‐old proband with resolved infantile spasms and normal development. Standard whole‐exome sequencing (WES) and Sanger sequencing were performed to identify the functional impact of the variant. Results: WES revealed a rare biallelic missense variant (c.3013G>C; p.Val1005Leu) in FCSK. RT‐qPCR showed a significant depletion in FCSK gene expression in the affected individual. Western blotting revealed reduced FCSK expression at the protein level compared to that in the control. Furthermore, 3D protein modeling suggested changes in the secondary structure, which might affect the overall FCSK protein function. Conclusion: This study broadens the mutation and phenotypic spectrum of FCSK‐associated developmental disorders. [ABSTRACT FROM AUTHOR]

Fucosylation is one of the most common modifications of oligo‐N‐acetyllactosamine (oligo‐LacNAc) glycans. However, none of known fucosyltransferases (FucTs) could install the α1,3‐linked fucose to the oligo‐LacNAc substrates in a site‐specific manner. Here, we report a facile and general redox‐controlled substrate engineering strategy for the site‐specific α1,3‐fucosylation of complex glycans containing multiple LacNAc units. This strategy takes advantage of an operationally simple oxidation enzyme module by using galactose oxidase (GOase) to convert the LacNAc unit into oxidized C6′‐aldehyde LacNAc sequence, which is not a good substrate for recombinant α1,3‐FucT from Helicobacter pylori strain 26695 (Hpα1,3FucT), enabling the site‐specific α1,3‐fucosylation at intact LacNAc sites. The general applicability and robustness of this strategy were demonstrated by the synthesis of a variety of structurally well‐defined fucosides of linear and branched O‐ and N‐linked glycans. [ABSTRACT FROM AUTHOR]

Core fucosylation, the attachment of α1,6‐fucose to the innermost N‐acetylglucosamine (GlcNAc) residue of N‐glycans, has a strong relationship with tumor growth, invasion, metastasis, prognosis, and immune evasion by regulating many membrane proteins. However, details about the functional mechanism are still largely unknown due to the lack of an effective analytical method to identify cell‐surface core‐fucosylated glycoproteins, and especially glycosylation sites. Here, we developed a sensitive and reversible labeling strategy for probing core fucosylation, by which core‐fucosylated glycoproteins that located on cell‐surface were selectively tagged by a biotinylated probe with high sensitivity. The labeled probe can be further broken enzymatically after the capture by affinity resin. The on‐bead traceless cleavage allowed the global mapping of core‐fucosylated glycoproteins and glycosylation sites by mass spectrometry (MS). The profile of core‐fucosylated glycoproteome provides an in‐depth understanding of the biological functions of core fucosylation. [ABSTRACT FROM AUTHOR]

Protein O‐fucosyltransferase 2 (PoFUT2) is an inverting glycosyltransferase (GT) that fucosylates thrombospondin repeats (TSRs) from group 1 and 2. PoFUT2 recognizes a large and diverse number of TSRs through a dynamic network of water‐mediated interactions. By X‐ray structural studies of C. elegans PoFUT2 complexed to a TSR of group 2, we demonstrate that this GT recognizes similarly the 3D structure of TSRs from both groups 1 and 2. Its active site is highly exposed to the solvent, suggesting that water molecules might also play an essential role in the fucosylation mechanism. We applied QM/MM methods using human PoFUT2 as a model, and found that HsPoFUT2 follows a classical SN2 reaction mechanism in which water molecules contribute to a great extent in facilitating the release of the leaving pyrophosphate unit, causing the H transfer from the acceptor nucleophile (Thr/Ser) to the catalytic base, which is the last event in the reaction. This demonstrates the importance of water molecules not only in recognition of the ligands but also in catalysis. [ABSTRACT FROM AUTHOR]

Some effector functions prompted by immunoglobulin G (IgG) antibodies, such as antibody‐dependent cell‐mediated cytotoxicity (ADCC), strongly depend on the N‐glycans linked to asparagine 297 of the Fc region of the protein. A single α‐(1,6)‐fucosyltransferase (FUT8) is responsible for catalyzing the addition of an α‐1,6‐linked fucose residue to the first GlcNAc residue of the N‐linked glycans. Antibodies missing this core fucose show a significantly enhanced ADCC and increased antitumor activity, which could help reduce therapeutic dose requirement, potentially translating into reduced safety concerns and manufacturing costs. Several approaches have been developed to modify glycans and improve the biological functions of antibodies. Here, we demonstrate that expression of a membrane‐associated anti‐FUT8 intrabody engineered to reside in the endoplasmic reticulum and Golgi apparatus can efficiently reduce FUT8 activity and therefore the core‐fucosylation of the Fc N‐glycan of an antibody. IgG1‐producing CHO cells expressing the intrabody secrete antibodies with reduced core fucosylation as demonstrated by lectin blot analysis and UPLC‐HILIC glycan analysis. Cells engineered to inhibit directly and specifically alpha‐(1,6)‐fucosyltransferase activity allows for the production of g/L levels of IgGs with strongly enhanced ADCC effector function, for which the level of fucosylation can be selected. The quick and efficient method described here should have broad practical applicability for the development of next‐generation therapeutic antibodies with enhanced effector functions. [ABSTRACT FROM AUTHOR]

Background Information: Like most other cell surface proteins, α5β1 integrin is glycosylated, which is required for its various activities in ways that mostly remain to be determined. Results: Here, we have established the first comprehensive site‐specific glycan map of α5β1 integrin that was purified from a natural source, that is, rat liver. This analysis revealed striking site selective variations in glycan composition. Complex bi, tri, or tetraantennary N‐glycans were predominant at various proportions at most potential N‐glycosylation sites. A few of these sites were nonglycosylated or contained high mannose or hybrid glycans, indicating that early N‐glycan processing was hindered. Almost all complex N‐glycans had fully galactosylated and sialylated antennae. Moderate levels of core fucosylation and high levels of O‐acetylation of NeuAc residues were observed at certain sites. An O‐linked HexNAc was found in an EGF‐like domain of β1 integrin. The extensive glycan information that results from our study was projected onto a map of α5β1 integrin that was obtained by homology modeling. We have used this model for the discussion of how glycosylation might be used in the functional cycle of α5β1 integrin. A striking example concerns the involvement of glycan‐binding galectins in the regulation of the molecular homeostasis of glycoproteins at the cell surface through the formation of lattices or endocytic pits according to the glycolipid‐lectin (GL‐Lect) hypothesis. Conclusion: We expect that the glycoproteomics data of the current study will serve as a resource for the exploration of structural mechanisms by which glycans control α5β1 integrin activity and endocytic trafficking. Significance: Glycosylation of α5β1 integrin has been implicated in multiple aspects of integrin function and structure. Yet, detailed knowledge of its glycosylation, notably the specific sites of glycosylation, is lacking. Furthermore, the α5β1 integrin preparation that was analyzed here is from a natural source, which is of importance as there is not a lot of literature in the field about the glycosylation of "native" glycoproteins. [ABSTRACT FROM AUTHOR]

Fucosyltransferases (FucTs) are crucial for the synthesis of Lewis‐type glycan epitopes. The synthetic capacity of efficient bacterial enzymes and their variants has not yet been fully exploited. In the present work, we investigated two previously described variants of α1,3FucT from Helicobacter pylori strains for their flexibility in substrate utilization and their applicability in the enzymatic synthesis of Lewis epitopes. We used the truncated enzyme variant of FutA from H. pylori 26695 (FucTΔ52A128N/H129E/Y132I/S46F, FucTΔ52‐4M) and the truncated α3FucT from H. pylori NCTC11639 (FucTΔ66). N‐Acetyllactosamine type 1 and type 2 as well as N',N"‐diacetyllactosamine were investigated as substrates. Both FucT variants exhibit α1,3/4FucT activity. Novel glycan structures were obtained displaying Lewis blood group antigens in a site‐specific sequence. Fucosylated N',N"‐diacetyllactosamine was synthesized for the first time with FucTΔ52‐4M. Our work paves the way for targeted fucosylation patterns that can be tested for lectin binding. [ABSTRACT FROM AUTHOR]

Fucosylation is essential for intercellular and intracellular recognition, cell‐cell interaction, fertilization, and inflammatory processes. Only five types of congenital disorders of glycosylation (CDG) related to an impaired fucosylation have been described to date: FUT8‐CDG, FCSK‐CDG, POFUT1‐CDG SLC35C1‐CDG, and the only recently described GFUS‐CDG. This review summarizes the clinical findings of all hitherto known 25 patients affected with those defects with regard to their pathophysiology and genotype. In addition, we describe five new patients with novel variants in the SLC35C1 gene. Furthermore, we discuss the efficacy of fucose therapy approaches within the different defects. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

FUT2, a protein that uses l‐fucose to mediate fucosylation of intestinal epithelial cells, is one of the detected gene variants in IBD patients. We aimed to investigate whether exogenous l‐fucose could be an enteral nutritional supplement to protect intestinal barrier function. The effect of l‐fucose on the restoration of epithelial barrier function in both the DSS‐induced colitis mouse model and LPS‐stimulated Caco‐2 cells was investigated, and the impact on fucosylation of epithelial cells was examined. The severity of DSS‐induced colitis was significantly reduced by l‐fucose. Restoration of epithelial barrier function by l‐fucose was detected. Direct l‐fucose‐mediated protection of tight junctions was observed in Caco‐2 cells. Moreover, exogenous l‐fucose promoted the exogenous metabolic pathway of l‐fucose, and fucosylation of epithelial cells both in vivo and in vitro. Moreover, knockout of the FUT2 gene restrained fucosylation and the protective effect of l‐fucose on barrier function. The severity of colitis was not improved by l‐fucose in Fut2 knockout mice. Therefore we conclude that exogenous l‐fucose protects intestinal barrier function and relieves intestinal inflammation via upregulation of FUT2‐mediated fucosylation of intestinal epithelial cells. [ABSTRACT FROM AUTHOR]

It is known that core‐type fucosylation is higher in prostate cancer cells than in other cancer cell types and is associated with high‐risk prostate cancer. Here, we developed an automated microcapillary electrophoresis‐based immunoassay system for measuring serum core‐type fucosylated prostate‐specific antigen (PSA) and evaluated whether the serum fucosylated PSA index (FPI) can detect high‐risk prostate cancer. Core‐type fucosylated‐free PSA was measured by our automated microcapillary electrophoresis‐based immunoassay system with Pholiota squarrosa lectin. The FPI was calculated from total PSA and the percentage of fucosylated‐free PSA. The optimum model to predict Gleason grade (GG) ≥2 was constructed by multivariate logistic regression analysis. Discrimination was assessed by determining the area under the receiver operator characteristic curve (AUC). The study included 252 men who underwent prostate needle biopsy due to elevated serum PSA levels (4‐20 ng/mL), including 138 with GG ≥2. A higher FPI was significantly associated with GG (P <.0001). Multivariate logistic regression analysis showed that age, prostate volume and FPI were significant predictors of GG ≥2. The AUC of FPI and the model were 0.729 (95% confidence interval [CI]: 0.668‐0.790) and 0.837 (95% CI: 0.788‐0.886), respectively, compared to 0.629 (95% CI: 0.561‐0.698) for PSA. Decision curve analysis showed the superior benefit of FPI and the model when compared to PSA. In a cohort with serum PSA levels <20 ng/mL, FPI could differentiate high‐risk prostate cancer from biopsy‐negative or low‐risk prostate cancer. Therefore, FPI could be a useful adjunct in prostate biopsy counseling for men with abnormal PSA levels. What's new The current prostate‐specific antigen (PSA) test lacks specificity and sensitivity for the accurate diagnosis of prostate cancer, leading to unnecessary biopsies and overdiagnosis. Previous studies have shown that core‐type fucosylation in prostate cancer cells is associated with high‐risk prostate cancer. Here, the authors developed an automated microcapillary electrophoresis‐based immunoassay system for measuring serum core‐type fucosylated free PSA. In a cohort with serum PSA levels <20 ng/ml, the fucosylated PSA index could differentiate high‐risk prostate cancer from biopsy‐negative or low‐risk prostate cancer. The fucosylated PSA index could be a useful adjunct in prostate biopsy counselling for men with abnormal PSA levels. [ABSTRACT FROM AUTHOR]

The deoxyhexose sugar L‐fucose is important for many biological processes within the human body and the associated microbiota. This carbohydrate is abundant in host gut mucosal surfaces, numerous microbial cell surface structures, and some dietary carbohydrates. Fucosylated oligosaccharides facilitate the establishment of a healthy microbiota and provide protection from infection. However, there are instances where pathogens can also exploit these fucosylated structures to cause infection. Furthermore, deficiencies in host fucosylation are associated with specific disease outcomes. This review focuses on our current understanding of the impact of fucosylation within the mucosal environment of the gastrointestinal tract with a specific emphasis on the mediatory effects in host–microbe interactions. [ABSTRACT FROM AUTHOR]

Aim: Nonalcoholic steatohepatitis (NASH) is a risk factor for nonvirus‐related hepatocellular carcinoma, which is increasing in prevalence. The aim of this study was to clarify the clinical application of fucosylated alpha‐fetoprotein (AFP‐L3) in the process of nonalcoholic fatty liver (NAFL) disease development. Methods: Serum samples from 115 diabetes mellitus (DM), 36 NAFL, and 119 NASH patients were analyzed for AFP‐L3 expression using raw data of a micro total analysis system. These data were then compared with the clinical characteristics of the patients. A validation study was also undertaken with 55 samples (17 NAFL and 38 NASH). Results: Trace amounts of AFP‐L3 were detected in 3.5%, 16.7%, and 58.0% of patients with DM, NAFL, and NASH, respectively. The odds ratio of AFP‐L3 positivity for the diagnosis of NASH in multivariate analysis was 9.81 (95% confidence interval, 3.77–25.5). The rates in patients without fibrosis or with stage 1, stage 2, stage 3, and stage 4 fibrosis were 14.7%, 31.3%, 63.0%, 86.2%, and 100%, respectively. The rates were significantly increased according to the advancement of liver fibrosis (p < 0.001); however, no difference in the positive rate of AFP‐L3 was observed between patients with and without fatty livers and between patients with normal and abnormal transaminase. The same relationship was also observed in the validation cohort. Conclusion: Abnormal fucosylation of AFP occurred in patients with NASH, so it could be useful for the screening of NASH in patients with DM, as well as for the differential diagnosis of NASH and the evaluation of fibrosis. [ABSTRACT FROM AUTHOR]

The reduction of antibody core‐fucosylation is known to enhance antibody‐dependent cellular cytotoxicity (ADCC). In this study, 5‐Thio‐l‐Fucose (ThioFuc) was investigated as a media and feed supplement for modulating the fucosylation profile of therapeutic proteins and, thereby, improving the resulting effector functions. Glycan analysis of five different therapeutic proteins produced by a diverse set of Chinese hamster ovary cell lines demonstrated a clone dependent impact of ThioFuc treatment. Using rituximab as a model, an efficient dose‐ and time‐dependent reduction of core‐fucosylation up to a minimum of 5% were obtained by ThioFuc. Besides a concomitant increase in the afucosylation level up to 48%, data also revealed up to 47% incorporation of ThioFuc in place of core‐fucosylation. In accordance with the glycan data, antibodies produced in the presence of ThioFuc revealed an enhanced FcγRIIIa binding up to 7.7‐fold. Furthermore, modified antibodies subjected to a cell‐based ADCC reporter bioassay proved to exert both a 1.5‐fold enhanced ADCC efficacy and 2.6‐fold enhancement in potency in comparison to their native counterparts—both of which contribute to an improvement in the ADCC activity. In conclusion, ThioFuc is a potent fucose derivative with potential applications in drug development processes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Rationale: Glycoprotein fucosylation, one of the major posttranslational modifications, is known to be highly involved in proteins related to various cancers. Fucosylation occurs in the core and/or outer sites of N-glycopeptides. Elucidation of the fucosylation type of N-glycoproteins is therefore important. However, it has remained a challenge to classify the fucosylation types of N-glycopeptides using collision-induced dissociation (CID) tandem mass (MS/MS) spectra. Methods: The relative intensities of theY1F, Y2F, Y3F, and Y4F product ions in the CIDMS/MS spectra of the IgG N-glycopeptides were measured for core fucosylation. The Core Fucose Index (CFI) was then calculated by multiplication of the relative intensities with a weight factor from logistic regression to differentiate between the core and none fucosylation. From the relative intensities of the B2F and B3SF ions of the MS/MS spectra of the AGP N-glycopeptides for outer fucosylation, the Outer Fucose Index (OFI) was calculated to differentiate between the outer and none fucosylation. Results: In order to classify core and/or outer fucosylation of N-glycoproteins, we defined the fucosylation score (F-score) by a sigmoidal equation using a combination of the CFI and the OFI. For application, we classified the fucosylation types of N-glycoproteins in human plasma with 99.7% accuracy from the F-score. Human plasma samples showed 54.4%, 33.3%, 10.3%, and 1.6% for none, core, outer, and dual fucosylated N-glycopeptides, respectively. Core fucosylation was abundant at mono- and bi-antennary N-glycopeptides. Outer fucosylation was abundant at tri- and tetra-antennary N-glycopeptides. In total, 113 N-glycopeptides of 29 glycoproteins from 3365 glycopeptide spectral matches (GPSMs) were classified for different types of fucosylation. Conclusions: We established an F-score to classify three different fucosylation types: core, outer, and dual types of N-glycopeptides. The fucosylation types of 20 new N-glycopeptides from 11 glycoproteins in human plasma were classified using the F-score. Therefore, the F-score can be useful for the automatic classification of different types of fucosylation in N-glycoproteins of biological fluids including plasma, serum, and urine. [ABSTRACT FROM AUTHOR]

Background &Aims: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer type with loco‐regional spread that makes the tumor surgically unresectable. Novel diagnostic tools are needed to improve detection of PDAC and increase patient survival. In this study we explore serum protein N‐glycan profiles from PDAC patients with regard to their applicability to serve as a disease biomarker panel. Methods: Total serum N‐glycome analysis was applied to a discovery set (86 PDAC cases/84 controls) followed by independent validation (26 cases/26 controls) using in‐house collected serum specimens. Protein N‐glycan profiles were obtained using ultrahigh resolution mass spectrometry and included linkage‐specific sialic acid information. N‐glycans were relatively quantified and case‐control classification performance was evaluated based on glycosylation traits such as branching, fucosylation, and sialylation. Results: In PDAC patients a higher level of branching (OR 6.19, P‐value 9.21 × 10−11) and (antenna)fucosylation (OR 13.27, P‐value 2.31 × 10−9) of N‐glycans was found. Furthermore, the ratio of α2,6‐ vs α2,3‐linked sialylation was higher in patients compared to healthy controls. A classification model built with three glycosylation traits was used for discovery (AUC 0.88) and independent validation (AUC 0.81), with sensitivity and specificity values of 0.85 and 0.71 for the discovery set and 0.75 and 0.72 for the validation set. Conclusion: Serum N‐glycome analysis revealed glycosylation differences that allow classification of PDAC patients from healthy controls. It was demonstrated that glycosylation traits rather than single N‐glycan structures obtained in this clinical glycomics study can serve as a basis for further development of a blood‐based diagnostic test. [ABSTRACT FROM AUTHOR]

As an immune checkpoint, programmed cell death 1 (PD‐1) and its ligand (PD‐L1) pathway plays a crucial role in CD8+ cytotoxic T lymphocytes (CTL) activation and provides antitumor responses. The N‐glycans of PD‐1 and PD‐L1 are highly core fucosylated, which are solely catalyzed by the core fucosyltransferase (Fut8). However, the precise biological mechanisms underlying effects of core fucosylation of PD‐1 and PD‐L1 on CTL activation have not been fully understood. In this study, we found that core fucosylation was significantly upregulated in lung adenocarcinoma. Compared to those of Fut8+/+OT‐I mice, the lung adenocarcinoma formation induced by urethane was markedly reduced in Fut8−/−OT‐I mice. De‐core fucosylation of PD‐1 compromised its expression on Fut8−/− CTL, resulted in enhanced Fut8−/− CTL activation and cytotoxicity, leading to more efficient tumor eradication. Indeed, loss of core fucosylation significantly enhanced the PD‐1 ubiquitination and in turn led to the degradation of PD‐1 in the proteasome. Our current work indicates that inhibition of core fucosylation is a unique strategy to reduce PD‐1 expression for the antilung adenocarcinoma immune therapy in the future. [ABSTRACT FROM AUTHOR]

Clear‐cell renal cell carcinoma (ccRCC) presents challenges to clinical management because of late‐stage detection, treatment resistance, and frequent disease recurrence. Metabolically, ccRCC has a well‐described Warburg effect utilization of glucose, but how this affects complex carbohydrate synthesis and alterations to protein and cell surface glycosylation is poorly defined. Using an imaging mass spectrometry approach, N‐glycosylation patterns and compositional differences were assessed between tumor and nontumor regions of formalin‐fixed clinical ccRCC specimens and tissue microarrays. Regions of normal kidney tissue samples were also evaluated for N‐linked glycan‐based distinctions between cortex, medullar, glomeruli, and proximal tubule features. Most notable was the proximal tubule localized detection of abundant multiantennary N‐glycans with bisecting N‐acetylglucosamine and multziple fucose residues. These glycans are absent in ccRCC tissues, while multiple tumor‐specific N‐glycans were detected with tri‐ and tetra‐antennary structures and varying levels of fucosylation and sialylation. A polycystic kidney disease tissue was also characterized for N‐glycan composition, with specific nonfucosylated glycans detected in the cyst fluid regions. Complementary to the imaging mass spectrometry analyses was an assessment of transcriptomic gene array data focused on the fucosyltransferase gene family and other glycosyltransferase genes. The transcript levels of the FUT3 and FUT6 genes responsible for the enzymes that add fucose to N‐glycan antennae were significantly decreased in all ccRCC tissues relative to matching nontumor tissues. These striking differences in glycosylation associated with ccRCC could lead to new mechanistic insight into the glycobiology underpinning kidney malignancies and suggest the potential for new therapeutic interventions and diagnostic markers. [ABSTRACT FROM AUTHOR]

Both Tamm‐Horsfall protein (THP) and collectin‐11 (CL‐11) are important molecules in acute kidney injury (AKI). In this study, we measured the change of glycosylation of THP in patients with AKI after surgery, using MALDI‐TOF MS and lectin array analysis. The amount of high‐mannose and core fucosylation in patients with AKI were higher than those in healthy controls. In vitro study showed that THP could bind to CL‐11 with affinity at 9.41 × 10−7 mol/L and inhibited activation of complement lectin pathway. The binding affinity decreased after removal of glycans on THP. Removal of fucose completely ablated the binding between the two proteins. While removal of high‐mannose or part of the N‐glycan decreased the binding ability to 30% or 60%. The results indicated that increase of fucose on THP played an important role via complement lectin pathway in AKI. [ABSTRACT FROM AUTHOR]

Objective: Aberrant glycosylation, mainly sialylation and fucosylation, is recently considered as a major hallmark of cancer. Aberrant sialylation has long been associated with various neoplastic diseases. However, role of aberrant sialylation in oral cancer is still in its infancy. The present study aimed to examine mRNA expressions of α-2, 3, α-2, 6 sialyltransferase (ST) families and sialidase in 160 human oral cancer tissues.Methods: mRNA expression of ST3GAL1, ST3GAL2, ST3GAL3, ST3GAL4, ST3GAL6, ST6GAL1, and neuraminidase 3 (NEU3) was analyzed by RT-qPCR in 80 paired malignant and adjacent normal tissues from oral cancer patients.Results: The results indicated significant (P ≤ .05) down-regulation of various STs (ST3GAL1, ST3GAL2, ST3GAL3, ST3GAL4, ST3GAL6, and ST6GAL1) and sialidases (NEU3) in malignant tissues as compared to adjacent normal tissues. Higher mRNA levels of ST3GAL2 and ST3GAL3 were significantly associated with advanced stage of the disease, lymph node involvement, and perineural invasion, which denote their role in progression and metastasis of oral cancer. Present study also revealed altered sialylation patterns according to anatomical site of the disease and tobacco habit.Conclusion: The study demonstrated significant role of elevated mRNA levels of ST3GAL2 and ST3GAL3 in disease progression and metastasis of oral carcinoma. [ABSTRACT FROM AUTHOR]

The canonical Wnt signalling pathway is a critical pathway involved in the proliferation of cells. It has been well‐established that it plays the central role during colorectal carcinogenesis and development. Yet the exact molecular mechanism of how the canonical Wnt pathway is fine‐tuned remains elusive. We found that SLC35C1, a GDP‐fucose transporter, negatively regulates the Wnt signalling pathway. We show here that SLC35C1 is reduced in all colon cancer by both immunohistochemistry images and TCGA data, whereas β‐catenin is increased. Down‐regulation of SLC35C1 is also detected by real‐time PCR in stage 3 and stage 4 colorectal cancer tissues. Moreover, analysing the TCGA database with cBioPortal reveals the negative correlation of SLC35C1 mRNA level to the expression of β‐catenin. Reduced SLC35C1 significantly promotes cell proliferation and colony formation of HEK293 cells. Meanwhile, in HEK293 cells silencing SLC35C1 activates canonical Wnt pathway, whereas overexpressing SLC35C1 inhibits it. Consistently, the reduction of SLC35C1 in HEK293 cells also elevated the mRNA level of Wnt target genes C‐myc, Axin2 and Cyclin D1, as well as the secretion of Wnt3a. In conclusion, we identified SLC35C1 as a negative regulator of the Wnt signalling pathway in colon cancer. Decreased SLC35C1 may cause over‐activation of Wnt signalling in colorectal cancer. [ABSTRACT FROM AUTHOR]

Objectives: Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infections in infants, and there is no vaccine available. In early life, the most important contributors to protection against infectious diseases are the innate immune response and maternal antibodies. However, antibody‐mediated protection against RSV disease is incompletely understood, as both antibody levels and neutralisation capacity correlate poorly with protection. Since antibodies also mediate natural killer (NK) cell activation, we investigated whether this functionality correlates with RSV disease. Methods: We performed an observational case–control study including infants hospitalised for RSV infection, hernia surgery or RSV‐negative respiratory viral infections. We determined RSV antigen‐specific antibody levels in plasma using a multiplex immunoassay. Subsequently, we measured the capacity of these antibodies to activate NK cells. Finally, we assessed Fc‐glycosylation of the RSV‐specific antibodies by mass spectrometry. Results: We found that RSV‐specific maternal antibodies activate NK cells in vitro. While concentrations of RSV‐specific antibodies did not differ between cases and controls, antibodies from infants hospitalised for severe respiratory infections (RSV and/or other) induced significantly less NK cell interferon‐γ production than those from uninfected controls. Furthermore, NK cell activation correlated with Fc‐fucosylation of RSV‐specific antibodies, but their glycosylation status did not significantly differ between cases and controls. Conclusion: Our results suggest that Fc‐dependent antibody function and quality, exemplified by NK cell activation and glycosylation, contribute to protection against severe RSV disease and warrant further studies to evaluate the potential of using these properties to evaluate and improve the efficacy of novel vaccines. [ABSTRACT FROM AUTHOR]

Summary: Forward genetic screens play a key role in the identification of genes contributing to plant stress tolerance. Using a screen for freezing sensitivity, we have identified a novel freezing tolerance gene, SENSITIVE‐TO‐FREEZING8, in Arabidopsis thaliana.We identified SFR8 using recombination‐based mapping and whole‐genome sequencing. As SFR8 was predicted to have an effect on cell wall composition, we used GC‐MS and polyacrylamide gel electrophoresis to measure cell‐wall fucose and boron (B)‐dependent dimerization of the cell‐wall pectic domain rhamnogalacturonan II (RGII) in planta. After treatments to promote borate‐bridging of RGII, we assessed freeze‐induced damage in wild‐type and sfr8 plants by measuring electrolyte leakage from freeze‐thawed leaf discs.We mapped the sfr8 mutation to MUR1, a gene encoding the fucose biosynthetic enzyme GDP‐d‐mannose‐4,6‐dehydratase. sfr8 cell walls exhibited low cell‐wall fucose levels and reduced RGII bridging. Freezing sensitivity of sfr8 mutants was ameliorated by B supplementation, which can restore RGII dimerization. B transport mutants with reduced RGII dimerization were also freezing‐sensitive.Our research identifies a role for the structure and composition of the plant primary cell wall in determining basal plant freezing tolerance and highlights the specific importance of fucosylation, most likely through its effect on the ability of RGII pectin to dimerize. [ABSTRACT FROM AUTHOR]

Background and Aim: Immunoglobulin subclass G4‐related disease (IgG4‐RD) is characterized by an abundance of IgG4 antibodies in the serum and tissue. Glycosylation status of antibodies can impact on immune effector functions and disease pathophysiology. We sought to establish glycosylation patterns in a prospective cohort of patients with IgG4‐RD and the relationship with disease activity and response to treatment. Methods: We assessed IgG Fc‐tail and Fab‐arm glycosylation status in patients with IgG4‐RD (n = 22), disease controls with primary sclerosing cholangitis (PSC) (n = 22), and healthy controls (n = 22). Serum IgG and subclasses were quantified using ELISA. Fc and Fab glycosylation were analyzed by mass spectrometry and lectin affinity chromatography, respectively. Disease activity, organ damage, and response to treatment were assessed using the IgG4 Responder Index. Results: Immunoglobulin G Fab sialylation was increased in IgG4‐RD compared with PSC and healthy control (P = 0.01), with a preferential increase in IgG4‐specific Fab sialylation, which was independent of IgG4 Fab‐arm exchange. There was a reduction in IgG1‐specific Fc bisection and hybrid structures in IgG4‐RD (P < 0.01), which recovered upon steroid treatment and correlated with disease activity. Overall, IgG Fc galactosylation was reduced in both IgG4‐RD and PSC (P < 0.01), with a preferential reduction in IgG1‐specific sialylation and enhancement of IgG4‐specific bisection in PSC. IgG4 fucosylation and IgG1/2/3 hybrid structures negatively correlated with complement C3 and C4 levels in IgG4‐RD (P < 0.01), but not PSC. Conclusion: We report the first study showing unique antibody glycosylation status in a prospective cohort of IgG4‐RD and PSC patients, which may determine modulation of the immune system and contribute to disease pathophysiology. [ABSTRACT FROM AUTHOR]

Dynamic turnover of cell‐surface glycans is involved in a myriad of biological events, making this process an attractive target for in vivo molecular imaging. Metabolic glycan labeling coupled with bioorthogonal chemistry has paved the way for visualizing glycans in living organisms. However, a two‐step labeling sequence is required, which suffers from the tissue‐penetration difficulties of the imaging probes. Here, by exploring the substrate promiscuity of endogenous glycosyltransferases, we developed a single‐step fluorescent glycan labeling strategy by using fluorophore‐tagged analogues of the nucleotide sugars. Injecting fluorophore‐tagged sialic acid and fucose into the yolk of zebrafish embryos at the one‐cell stage enables systematic imaging of sialylation and fucosylation in live zebrafish embryos at distinct developmental stages. From these studies, we obtained insights into the role of sialylated and fucosylated glycans in zebrafish hematopoiesis. [ABSTRACT FROM AUTHOR]

We have used tandem mass spectrometry (MS/MS)‐based analysis of glycopeptides in order to identify the composition and structure of rare glycoforms. The results illustrate utility of low‐energy MS/MS for structure identification. We have shown the presence of bifucosylated and trifucosylated glycoforms in human α‐1‐acid glycoprotein (AGP), a major plasma glycoprotein. Fucosylation in the case of AGP always occurs on the antennae; core fucosylation was not observed. [ABSTRACT FROM AUTHOR]

We describe a chemoenzymatic strategy that can give a library of differentially fucosylated and sialylated oligosaccharides starting from a single chemically synthesized tri‐N‐acetyllactosamine derivative. The common precursor could easily be converted into 6 different hexasaccharides in which the glucosamine moieties are either acetylated (GlcNAc) or modified as a free amine (GlcNH2) or Boc (GlcNHBoc). Fucosylation of the resulting compounds by a recombinant fucosyl transferase resulted in only modification of the natural GlcNAc moieties, providing access to 6 selectively mono‐ and bis‐fucosylated oligosaccharides. Conversion of the GlcNH2 or GlcNHBoc moieties into the natural GlcNAc, followed by sialylation by sialyl transferases gave 12 differently fucosylated and sialylated compounds. The oligosaccharides were printed as a microarray that was probed by several glycan‐binding proteins, demonstrating that complex patterns of fucosylation can modulate glycan recognition. [ABSTRACT FROM AUTHOR]

A straightforward glycosylation method is described to regio‐ and stereoselectively introduce two α‐l‐fucose moieties directly to the secondary rim of β‐cyclodextrin. Using NMR and MS fragmentation studies, the nonasaccharide structure was determined, which was also visualized using molecular dynamics simulations. The reported glycosylation method proved to be robust on gram‐scale, and may be generally applied to directly glycosylate β‐cyclodextrins to make well‐defined multivalent glycoclusters. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) is the third most‐common cause of cancer‐related death worldwide. Most cases of HCC develop in patients that already have liver cirrhosis and have been recommended for surveillance for an early onset of HCC. Cirrhosis is the final common pathway for several etiologies of liver disease, including hepatitis B and C, alcohol, and increasingly non‐alcoholic fatty liver disease. Only 20–30% of patients with HCC are eligible for curative therapy due primarily to inadequate early‐detection strategies. Reliable, accurate biomarkers for HCC early detection provide the highest likelihood of curative therapy and survival; however, current early‐detection methods that use abdominal ultrasound and serum alpha fetoprotein are inadequate due to poor adherence and limited sensitivity and specificity. There is an urgent need for convenient and highly accurate validated biomarkers for HCC early detection. The theme of this review is the development of new methods to discover glycoprotein‐based markers for detection of HCC with mass spectrometry approaches. We outline the non‐mass spectrometry based methods that have been used to discover HCC markers including immunoassays, capillary electrophoresis, 2‐D gel electrophoresis, and lectin‐FLISA assays. We describe the development and results of mass spectrometry‐based assays for glycan screening based on either MALDI‐MS or ESI analysis. These analyses might be based on the glycan content of serum or on glycan screening for target molecules from serum. We describe some of the specific markers that have been developed as a result, including for proteins such as Haptoglobin, Hemopexin, Kininogen, and others. We discuss the potential role for other technologies, including PGC chromatography and ion mobility, to separate isoforms of glycan markers. Analyses of glycopeptides based on new technologies and innovative softwares are described and also their potential role in discovery of markers of HCC. These technologies include new fragmentation methods such as EThcD and stepped HCD, which can identify large numbers of glycopeptide structures from serum. The key role of lectin extraction in various assays for intact glycopeptides or their truncated versions is also described, where various core‐fucosylated and hyperfucosylated glycopeptides have been identified as potential markers of HCC. Finally, we describe the role of LC‐MRMs or lectin‐FLISA MRMs as a means to validate these glycoprotein markers from patient samples. These technological advancements in mass spectrometry have the potential to lead to novel biomarkers to improve the early detection of HCC. [ABSTRACT FROM AUTHOR]

Summary: 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 PstDC3000. 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 pattern‐triggered immunity and effector‐triggered immunity, including glycosylation of pattern‐recognition 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. [ABSTRACT FROM AUTHOR]

The remarkable pro‐apoptotic properties of tumour necrosis factor (TNF)‐related apoptosis‐inducing ligand (TRAIL) have led to considerable interest in this protein as a potential anticancer therapeutic. However, TRAIL is largely ineffective in inducing apoptosis in certain cancer cells, and the mechanisms underlying this selectivity are unknown. In colon adenocarcinomas, posttranslational modifications including O‐ and N‐ glycosylation of death receptors were found to correlate with TRAIL‐induced apoptosis. Additionally, mRNA levels of fucosyltransferase 3 (FUT3) and 6 (FUT6) were found to be high in the TRAIL‐sensitive colon adenocarcinoma cell line COLO 205. In this study, we use agonistic receptor‐specific TRAIL variants to dissect the contribution of FUT3 and FUT6‐mediated fucosylation to TRAIL‐induced apoptosis via its two death receptors, DR4 and DR5. Triggering of apoptosis by TRAIL revealed that the low FUT3/6‐expressing cells DLD‐1 and HCT 116 are insensitive to DR5 but not to DR4‐mediated apoptosis. By contrast, efficient apoptosis is mediated via both receptors in high FUT3/6‐expressing COLO 205 cells. The reconstitution of FUT3/6 expression in DR5‐resistant cells completely restored TRAIL sensitivity via this receptor, while only marginally enhancing apoptosis via DR4 at lower TRAIL concentrations. Interestingly, we observed that induction of the salvage pathway by external administration of l‐fucose restores DR5‐mediated apoptosis in both DLD‐1 and HCT 116 cells. Finally, we show that fucosylation influences the ligand‐independent receptor association that leads to increased death inducing signalling complex (DISC) formation and caspase‐8 activation. Taken together, these results provide evidence for the differential impact of fucosylation on signalling via DR4 or DR5. These findings provide novel opportunities to enhance TRAIL sensitivity in colon adenocarcinoma cells that are highly resistant to DR5‐mediated apoptosis. Because of its ability to induce apoptosis in tumour cells, TRAIL [Tumour necrosis factor (TNF)‐related apoptosis‐inducing ligand] has sparked a lot of interest in oncology research. Posttranslational modifications and upregulation of fucosylation were previously found to correlate with TRAIL‐induced apoptosis. This study shows that fucosylation triggers the clustering of death receptors DR4 and DR5, leading to increased cell death. Moreover, apoptosis mediated by DR5 can be stimulated by simple external administration of l‐fucose. [ABSTRACT FROM AUTHOR]

Rationale: Certain lung cancer patients express elevated Fucosyl Monosialoganglioside (Fuc‐GM1) in circulation compared to control groups. Several sensitive methods involving characterization of Fuc‐GM1 have been reported. However, a highly specific and sensitive method for quantifying multiple potential Fuc‐GM1 biomarkers present in various biological matrices has not been reported to date. Methods: Individual Fuc‐GM1 analogs in a commercially obtained standard mixture were characterized using HPLC/UV/MS and high‐resolution mass spectrometry (HRMS). Proprietary antibodies, mAb1 and mAb2, were used to selectively capture and pre‐concentrate the soluble and drug‐bound forms of Fuc‐GM1 molecules present in human serum and whole blood, eliminating the background matrix components. Immunocapture extraction (ICE) followed by HPLC/MS/MS was used to quantify specific Fuc‐GM1 analogs in biological matrices. Results: The concentration of individual Fuc‐GM1 analogs in the standard mixture was estimated to be 7–34%, using HPLC/UV/MS. Using the standard mixture spiked into the biological matrices (100 μL), the lower limit of quantification (LLOQ) of each analog was 0.2–0.4 ng/mL with a dynamic range of up to 200 ng/mL. The applicability of the ICE‐HPLC/MS/MS method was demonstrated by detecting endogenous Fuc‐GM1 analogs present in rat blood and in several lung cancer cell lines. Conclusions: This highly specific and sensitive HPLC/MS/MS method for quantifying individual potential Fuc‐GM1 biomarkers in serum and whole blood can play a critical role in patient stratification strategies and during drug treatment. This method can be employed for monitoring both free (soluble) form and antibody drug‐bound Fuc‐GM1. [ABSTRACT FROM AUTHOR]

Abstract: Enzymatic synthesis of para‐lacto‐N‐hexaose and its isomeric structures as well as those α1,2‐fucosylated variants naturally occurring in human milk oligosaccharide (HMOs) was achieved using a sequential one‐pot enzymatic system. Three glycosylation routes comprising bacterial glycosyltransferases and corresponding sugar‐nucleotide‐generating enzymes were developed to facilitate efficient production of extended type‐1 and type‐2 N‐acetyllactosamine (LacNAc) backbones and hybrid chains. Further fucosylation efficiency of two α1,2‐fucosyltransferases on both type‐1 and type‐2 chains of the hexasaccharide was investigated to achieve practical synthesis of the fucosylated glycans. The availability of structurally defined HMOs offers a practical approach for investigating future biological applications. [ABSTRACT FROM AUTHOR]

Protein O‐fucosylation is an important post‐translational modification (PTM) found in cysteine‐rich repeats in proteins. Protein O‐fucosyltransferases 1 and 2 (PoFUT1 and PoFUT2) are the enzymes responsible for this PTM and selectively glycosylate specific residues in epidermal growth factor‐like (EGF) repeats and thrombospondin type I repeats (TSRs), respectively. Within the past six years, crystal structures of both enzymes have been reported, revealing important information on how they recognize protein substrates and achieve catalysis. Here, the structural information available today is summarized and how PoFUT1 and PoFUT2 employ different catalytic mechanisms is discussed. [ABSTRACT FROM AUTHOR]

Breast cancer tissue overexpresses fucosylated glycans, such as sialyl‐Lewis X/A (sLeX/A), and α‐1,3/4‐fucosyltransferases (FUTs) in relation to increased disease progression and metastasis. These glycans in tumor circulating cells mediate binding to vascular E‐selectin, initiating tumor extravasation. However, their role in breast carcinogenesis is still unknown. Here, we aimed to define the contribution of the fucosylated structures, including sLeX/A, to cell adhesion, cell signaling, and cell proliferation in invasive ductal carcinomas (IDC), the most frequent type of breast cancer. We first analyzed expression of E‐selectin ligands in IDC tissue and established primary cell cultures from the tissue. We observed strong reactivity with E‐selectin and anti‐sLeX/A antibodies in both IDC tissue and cell lines, and expression of α‐1,3/4 FUTs FUT4, FUT5, FUT6, FUT10, and FUT11. To further assess the role of fucosylation in IDC biology, we immortalized a primary IDC cell line with human telomerase reverse transcriptase to create the ‘CF1_T cell line’. Treatment with 2‐fluorofucose (2‐FF), a fucosylation inhibitor, completely abrogated its sLeX/A expression and dramatically reduced adherence of CF1_T cells to E‐selectin under hemodynamic flow conditions. In addition, 2‐FF‐treated CF1_T cells showed a reduced migratory ability, as well as decreased cell proliferation rate. Notably, 2‐FF treatment lowered the growth factor expression of CF1_T cells, prominently for FGF2, vascular endothelial growth factor, and transforming growth factor beta, and negatively affected activation of signal‐regulating protein kinases 1 and 2 and p38 mitogen‐activated protein kinase signaling pathways. These data indicate that fucosylation licenses several malignant features of IDC, such as cell adhesion, migration, proliferation, and growth factor expression, contributing to tumor progression. [ABSTRACT FROM AUTHOR]

Abstract: Fucosylated chondroitin sulfate (FuCS) is a structurally distinct glycosaminoglycan with excellent anticoagulant activity. Studies show that FuCS and its depolymerized fragments exhibit a different anticoagulant mechanism from that of heparin derivatives, with decreased risks of adverse effects and bleeding. However, further exploitation has been hindered by the scarcity of structurally defined oligosaccharides. Herein, facile method is reported for the synthesis of the repeating trisaccharide unit of FuCS based on the degradation of chondroitin sulfate polymers. A series of simplified FuCS glycomimetics that have highly tunable structures, controllable branches, and defined sulfation motifs were generated by copper‐catalyzed alkyne–azide cycloaddition. Remarkable improvement in activated partial thromboplastin time (APTT) assay activities was observed as the branches increased, but no significant influences were observed for prothrombin time (PT) and thrombin time (TT) assay activities. Further FXase inhibition tests suggested that glycoclusters 33 b–40 b selectively inhibited intrinsic anticoagulant activities, but had little effect on the extrinsic and common coagulation pathways. Notably, glycoclusters with the 2,4‐di‐O‐sulfated fucosyl residue displayed the most potency, which was in consistent with that of natural polysaccharides. These FuCS clusters demonstrated potency to mimic linear glycosaminoglycans and offer a new framework for the development of novel anticoagulant agents. [ABSTRACT FROM AUTHOR]

Abstract: Glycans anchored on cell‐surface receptors are active modulators of receptor signaling. A strategy is presented that enforces transient changes to cell‐surface glycosylation patterns to tune receptor signaling. This approach, termed in situ glycan editing, exploits recombinant glycosyltransferases to incorporate monosaccharides with linkage specificity onto receptors in situ. α2,3‐linked sialic acid or α1,3‐linked fucose added in situ suppresses signaling through epidermal growth factor receptor and fibroblast growth factor receptor. We also applied the same strategy to regulate the electrical signaling of a potassium ion channel–human ether‐à‐go‐go‐related gene channel. Compared to gene editing, no long‐term perturbations are introduced to the treated cells. In situ glycan editing therefore offers a promising approach for studying the dynamic role of specific glycans in membrane receptor signaling and ion channel functions. [ABSTRACT FROM AUTHOR]

The mechanistic underpinnings of the complex process of plant polysaccharide biosynthesis are poorly understood, largely because of the resistance of glycosyltransferase (GT) enzymes to structural characterization. In Arabidopsis thaliana, a glycosyl transferase family 37 (GT37) fucosyltransferase 1 ( AtFUT1) catalyzes the regiospecific transfer of terminal 1,2-fucosyl residues to xyloglucan side chains - a key step in the biosynthesis of fucosylated sidechains of galactoxyloglucan. We unravel the mechanistic basis for fucosylation by AtFUT1 with a multipronged approach involving protein expression, X-ray crystallography, mutagenesis experiments and molecular simulations. Mammalian cell culture expressions enable the sufficient production of the enzyme for X-ray crystallography, which reveals the structural architecture of AtFUT1 in complex with bound donor and acceptor substrate analogs. The lack of an appropriately positioned active site residue as a catalytic base leads us to propose an atypical water-mediated fucosylation mechanism facilitated by an H-bonded network, which is corroborated by mutagenesis experiments as well as detailed atomistic simulations. [ABSTRACT FROM AUTHOR]

ABSTRACT During antibody dependent cell cytotoxicity (ADCC) the target cells are killed by monocytes and natural killer cells. ADCC is enhanced when the antibody heavy chain's core N-linked glycan lacks the fucose molecule(s). Several strategies have been utilized to generate fully afucosylated antibodies. A commonly used and efficient approach has been knocking out the FUT8 gene of the Chinese hamster ovary (CHO) host cells, which results in expression of antibody molecules with fully afucosylated glycans. However, a major drawback of the FUT8-KO host is the requirement for undertaking two separate cell line development (CLD) efforts in order to obtain both primarily fucosylated and fully afucosylated antibody species for comparative studies in vitro and in vivo. Even more challenging is obtaining primarily fucosylated and FUT8-KO clones with similar enough product quality attributes to ensure that any observed ADCC advantage(s) can be strictly attributed to afucosylation. Here, we report generation and use of a FX knockout (FXKO) CHO host cell line that is capable of expressing antibody molecules with either primarily fucosylated or fully afucosylated glycan profiles with otherwise similar product quality attributes, depending on addition of fucose to the cell culture media. Hence, the FXKO host not only obviates the requirement for undertaking two separate CLD efforts, but it also averts the need for screening many colonies to identify clones with comparable product qualities. Finally, FXKO clones can express antibodies with the desired ratio of primarily fucosylated to afucosylated glycans when fucose is titrated into the production media, to allow achieving intended levels of FcγRIII-binding and ADCC for an antibody. Biotechnol. Bioeng. 2017;114: 632-644. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Fucosylated chondroitin sulfate (fCS)-a glycosaminoglycan (GAG) found in sea cucumbers-has recently attracted much attention owing to its biological properties. In particular, a low molecular mass fCS polysaccharide has very recently been suggested as a strong candidate for the development of an antithrombotic drug that would be safer and more effective than heparin. To avoid the use of animal sourced drugs, here we present the chemical transformation of a microbial sourced unsulfated chondroitin polysaccharide into a small library of fucosylated (and sulfated) derivatives thereof. To this aim, a modular approach based on the different combination of only five reactions was employed, with an almost unprecedented polysaccharide branching by O-glycosylation as the key step. The library was differentiated for sulfation patterns and/or positions of the fucose branches, as confirmed by detailed 2D NMR spectroscopic analysis. These semi-synthetic polysaccharides will allow a wider and more accurate structure-activity relationship study with respect to those reported in literature to date. [ABSTRACT FROM AUTHOR]