Search

Background: Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor. Notwithstanding tremendous efforts having been put in multi-omics research to profile the dysregulated molecular mechanisms and cellular pathways, there is still a lack of understanding about the glycoproteomic of GBM. Glycosylation as one of the most important post-translational modifications is crucial in regulating cell proliferation and relevant oncogenic pathways. Results: In the study, we systematically profiled N-glycoproteomics of para-cancerous and cancerous tissues from GBM patients to reveal the site-specific N-glycosylation pattern defined by intact glycopeptides. We identified and quantified 1863 distinct intact glycopeptides (IGPs) with 161 N-linked glycan compositions and 326 glycosites. There were 396 IGPs from 43 glycoproteins differed between adjacent tissues and GBM. Then, proteomic and glycoproteomic data were combined, and the normalized glycosylation alteration was calculated to determine whether the difference was attributed to the global protein levels or glycosylation. The altered glycosylation triggered by site-specific N-glycans and glycoprotein abundance, as well as glycosite heterogeneity, were demonstrated. Ultimately, an examination of the overall glycosylation levels revealed a positive contribution of sialylated or/and fucosylated glycans. Conclusions: Overall, the dataset highlighted molecular complexity and distinct profiling at translational and post-translational levels, providing valuable information for novel therapeutic approaches and specific detection strategies. [ABSTRACT FROM AUTHOR]

Immune checkpoint blockade therapy, represented by anti-PD-1/PD-L1 monoclonal antibodies, has significantly changed the immunotherapy landscape. However, the treatment is still limited by unsatisfactory response rates, immune-related adverse effects, and drug resistance. Current studies have established that glycosylation, a common post-translational modification, is crucial in promoting cancer progression and immune invasion. Targeting aberrant glycosylation in cancers presents precision medicine regimens for monitoring cancer progression and developing personalized medicine. Notably, the immune checkpoints PD-1 and PD-L1 are highly glycosylated, which affects PD-1/PD-L1 interaction and the binding of anti-PD-1/PD-L1 monoclonal antibodies. Recent achievements in glycoscience to enhance patient outcomes, referred to as glycotherapy, have underscored their high potency in advancing PD-1/PD-L1 blockade therapies, i.e., glycoengineered antibodies with improved binding toward PD-1/PD-L1, pharmaceutic inhibitors for core fucosylation and sialylation, and synergistic treatment with the antibody–sialidase conjugate. This review briefly introduces the PD-1/PD-L1 axis and glycosylation and highlights the fundamental and applied advances in glycoscience that improve PD-1/PD-L1 immunoblockade therapies. [ABSTRACT FROM AUTHOR]

Antibody glycosylation patterns can affect antibody functionality and thereby contribute to protection against invading pathogens. During pregnancy, maternal antibodies can be transferred through the placenta and contribute to modulating both the mother's and her child's immune responses. Although several studies of IgG glycosylation during pregnancy have been carried out, very few cohorts studied were from sub-Saharan Africa, where exposure to microorganisms and parasites is high. In Lambaréné, Gabon, 106 pregnant women in their third trimester were enrolled into this study. At enrolment, urine, stool, and blood samples were collected from the mothers to assess Schistosoma haematobium (S. haematobium), Plasmodium falciparum (P. falciparum) and other parasite infections. During delivery, cord blood samples were collected. The children were followed, and blood samples were collected at 9 and 12 months of age. IgG Fc glycosylation was measured by liquid chromatography-mass spectrometry, determining fucosylation, galactosylation, sialylation, bisection, and sialylation per galactose (SA/gal). Among the 106 pregnant women, 33 (31%) were infected by at least one parasite. The antibody glycosylation patterns in maternal and cord blood showed distinct profiles when compared to that of infants at 9 and 12 months. IgG galactosylation was higher in maternal/cord blood, while fucosylated IgG was higher in children up to 1 year of age. Maternal parasitic infection was associated with lower IgG2 and IgG3/IgG4 galactosylation in cord blood and lower IgG3/IgG4 galactosylation in children. When maternal IgG galactosylation and, consequently, cord blood were categorized as high, children at 9 and 12 months of age showed higher IgG galactosylation compared to children of mothers with low IgG galactosylation. As IgG Fc galactosylation can have functional consequences, it might provide valuable information for developing effective preventive and treatment strategies for vulnerable populations. [ABSTRACT FROM AUTHOR]

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]

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]

The LebLea heptasaccharide is a tumor-associated carbohydrate antigen that was isolated from the human colonic adenocarcinoma cell line Colo205 and is a good target for the development of anti-cancer immunotherapeutics. However, it displays on its reducing end the Leb tetrasaccharide: α-l-Fucp-(1→2)-β-d-Galp-(1→3)-[α-l-Fucp-(1→4)]-d-GlcNAcp and the H-type 1 (H-1 antigen) trisaccharide: α-l-Fucp-(1→2)-β-d-Galp-(1→3)-d-GlcNAcp that are also found on noncancerous tissues. To discover analogues or fragments of LebLea that could be used as immunotherapeutics while not triggering immune responses against Leb and the H-1 antigen, we have synthesized the Leb tetrasaccharide hexyl glycoside and the Leb and H-1 antigens aminohexyl glycosides to be used in ELISA experiments. We describe an improved preparation of the 6-O-benzyl-2,3,4-tri-O-acetyl-α-d-galactopyranosyl bromide in neutral conditions and demonstrate the importance of appropriately "matching" the reactivity of acceptors with that of glycosyl donors. Mono- and di-fucosylation of a disaccharide diol acceptor with per-benzylated thioethyl fucoside activated in situ with bromine and under halide ion catalysis is described and our results are compared to literature reports. We observed that our fucosylation reactions required higher equivalents of fucosyl donor and extended reaction times than previously reported. We propose that the protecting groups on the galactosyl unit led to a reduced reactivity of the acceptor. The protected intermediates were converted to 6-azido hexyl glycosides and submitted to dissolving metal conditions to give 6-aminohexyl glycosides. We also prepared the n-hexyl tetrasaccharide glycoside Leb that will be used as a soluble antigen in competitive ELISA experiments. [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]

N-linked glycoproteins are rich in seminal plasma, playing essential roles in supporting sperm function and fertilization process. The alteration of seminal plasma glycans and its correspond glycoproteins may lead to sperm dysfunction and even infertility. In present study, an integrative analysis of glycoproteomic and proteomic was performed to investigate the changes of site-specific glycans and glycoptoteins in seminal plasma of asthenozoospermia. By large scale profiling and quantifying 5,018 intact N- glycopeptides in seminal plasma, we identified 92 intact N- glycopeptides from 34 glycoproteins changed in asthenozoospermia. Especially, fucosylated glycans containing lewis x, lewis y and core fucosylation were significantly up-regulated in asthenozoospermia compared to healthy donors. The up-regulation of fucosylated glycans in seminal plasma may interfere sperm surface compositions and regulation of immune response, which subsequently disrupts sperm function. Three differentiated expression of seminal vesicle-specific glycoproteins (fibronectin, seminogelin-2, and glycodelin) were also detected with fucosylation alteration in seminal plasma of asthenozoospermia. The interpretation of the altered site-specific glycan structures provides data for the diagnosis and etiology analysis of male infertility, as well as providing new insights into the potential therapeutic targets for male infertility. [ABSTRACT FROM AUTHOR]

Higher breast cancer mortality rates continue to disproportionally affect black women (BW) compared to white women (WW). This disparity is largely due to differences in tumor aggressiveness that can be related to distinct ancestry-associated breast tumor microenvironments (TMEs). Yet, characterization of the normal microenvironment (NME) in breast tissue and how they associate with breast cancer risk factors remains unknown. N-glycans, a glucose metabolism-linked post-translational modification, has not been characterized in normal breast tissue. We hypothesized that normal female breast tissue with distinct Breast Imaging and Reporting Data Systems (BI-RADS) categories have unique microenvironments based on N-glycan signatures that varies with genetic ancestries. Profiles of N-glycans were characterized in normal breast tissue from BW (n  = 20) and WW (n  = 20) at risk for breast cancer using matrix assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI). A total of 176 N-glycans (32 core-fucosylated and 144 noncore-fucosylated) were identified in the NME. We found that certain core-fucosylated, outer-arm fucosylated and high-mannose N-glycan structures had specific intensity patterns and histological distributions in the breast NME dependent on BI-RADS densities and ancestry. Normal breast tissue from BW, and not WW, with heterogeneously dense breast densities followed high-mannose patterns as seen in invasive ductal and lobular carcinomas. Lastly, lifestyles factors (e.g. age, menopausal status, Gail score, BMI, BI-RADS) differentially associated with fucosylated and high-mannose N-glycans based on ancestry. This study aims to decipher the molecular signatures in the breast NME from distinct ancestries towards improving the overall disparities in breast cancer burden. [ABSTRACT FROM AUTHOR]

Simple Summary: Cancer is a difficult-to-cure disease with high worldwide incidence and mortality. Among the many changes observed in cancer cells and patient samples is altered glycosylation, a commonly observed modification of biomolecules such as proteins. These glycan structures can dictate protein function, and dysregulation of glycosylation can contribute to tumor migration and metastasis. Thus, manipulation of glycosylation states may be a novel approach to cancer treatment. One target of the well-known tumor suppressor p53 is FUCA1, encoding alpha-L-fucosidase, which plays a role in glycosylation, although the exact mechanism linking FUCA1 to cancer is unclear. Investigation into these glycosylation processes and the mechanisms linking the p53-FUCA1 axis to cancer development may provide new insights into this disease and suggest new drug targets for cancer therapies. Cancer is a difficult-to-cure disease with high worldwide incidence and mortality, in large part due to drug resistance and disease relapse. Glycosylation, which is a common modification of cellular biomolecules, was discovered decades ago and has been of interest in cancer research due to its ability to influence cellular function and to promote carcinogenesis. A variety of glycosylation types and structures regulate the function of biomolecules and are potential targets for investigating and treating cancer. The link between glycosylation and carcinogenesis has been more recently revealed by the role of p53 in energy metabolism, including the p53 target gene alpha-L-fucosidase 1 (FUCA1), which plays an essential role in fucosylation. In this review, we summarize roles of glycan structures and glycosylation-related enzymes to cancer development. The interplay between glycosylation and tumor microenvironmental factors is also discussed, together with involvement of glycosylation in well-characterized cancer-promoting mechanisms, such as the epidermal growth factor receptor (EGFR), phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) and p53-mediated pathways. Glycan structures also modulate cell–matrix interactions, cell–cell adhesion as well as cell migration and settlement, dysfunction of which can contribute to cancer. Thus, further investigation of the mechanistic relationships among glycosylation, related enzymes and cancer progression may provide insights into potential novel cancer treatments. [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]

The fucosylation of glycoproteins regulates diverse physiological processes. Inhibitors that can control cellular levels of protein fucosylation have consequently emerged as being of high interest. One area where inhibitors of fucosylation have gained significant attention is in the production of afucosylated antibodies, which exhibit superior antibody-dependent cell cytotoxicity as compared to their fucosylated counterparts. Here, we describe β-carbafucose, a fucose derivative in which the endocyclic ring oxygen is replaced by a methylene group, and show that it acts as a potent metabolic inhibitor within cells to antagonize protein fucosylation. β-carbafucose is assimilated by the fucose salvage pathway to form GDP-carbafucose which, due to its being unable to form the oxocarbenium ion-like transition states used by fucosyltransferases, is an incompetent substrate for these enzymes. β-carbafucose treatment of a CHO cell line used for high-level production of the therapeutic antibody Herceptin leads to dose-dependent reductions in core fucosylation without affecting cell growth or antibody production. Mass spectrometry analyses of the intact antibody and N-glycans show that β-carbafucose is not incorporated into the antibody N-glycans at detectable levels. We expect that β-carbafucose will serve as a useful research tool for the community and may find immediate application for the rapid production of afucosylated antibodies for therapeutic purposes. [ABSTRACT FROM AUTHOR]

Copyright of Oncolog-Hematolog is the property of MEDICHUB MEDIA, S.R.L. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

A range of recombinant monoclonal antibodies (rMAbs) have found application in treating diverse diseases, spanning various cancers and immune system disorders. Chinese hamster ovary (CHO) cells have emerged as the predominant choice for producing these rMAbs due to their robustness, ease of transfection, and capacity for posttranslational modifications akin to those in human cells. Transient transfection and/or stable expression could be conducted to express rMAbs in CHO cells. To bolster the yield of rMAbs in CHO cells, a multitude of approaches have been developed, encompassing vector optimization, medium formulation, cultivation parameters, and cell engineering. This review succinctly outlines these methodologies when also addressing challenges encountered in the production process, such as issues with aggregation and fucosylation. [ABSTRACT FROM AUTHOR]

Background: Gastric cancer (GC) is a malignant digestive tract tumor with a high recurrence rate and poor prognosis. Fucosylation is important in tumor glycosylation, in which the key enzyme is fucosyltransferase (FUT). FUT11 is a member of the fucosyltransferase family and has been closely associated with the development of multiple cancers. However, the specific relationship between FUT11 and GC prognosis and its molecular mechanism has not been fully studied. This study explored FUT11 expression, clinical correlation, and its role in GC occurrence and development to deepen understanding of its function. Methods: FUT11 expression in 33 cancers was preliminarily analyzed using the Tumor Immunoassay Resource (TIMER2.0) database. FUT11 expression in GC was evaluated using The Cancer Genome Atlas stomach adenocarcinoma (TCGA-STAD) and Gene Expression Profiling Interactive Analysis (GEPIA2) data and verified using the Gene Expression Omnibus (GEO) GSE65801 dataset. Furthermore, we studied the survival prognosis of FUT11 in GC and analyzed its effect on the survival rate of patients with GC using the KM-plotter. We also performed COX regression analysis on TCGA GC clinical data and analyzed FUT11 expression in the pathway using the STRING and LinkedOmics databases. Moreover, the relationship between FUT11 and GC immune infiltration level was examined, and the Kaplan–Meier survival analysis diagram was constructed. The FUT11 genetic variation information was retrieved using cBioPortal, and its drug sensitivity was analyzed using CellMiner. Finally, differential FUT11 expression in GC tissues was verified using immunohistochemistry. Results: The data mining and analysis demonstrated that FUT11 expression was abnormally elevated in GC tissues and correlated with poor patient prognosis. The FUT11 expression level was an independent prognostic factor for GC. The difference in FUT11 expression level resulted in different degrees of immune cell infiltration in the patients with GC, which might regulate the tumor microenvironment. FUT11 affected GC development by participating in cancer pathways such as PI3K–AKT, neuroactive ligand–receptor, and MAPK. Immunohistochemical staining revealed that FUT11 was highly expressed in GC. Conclusions: This study revealed that FUT11 expression is significantly increased in GC tissues. This increase is associated with poor prognosis and might affect immune regulation. FUT11 might have immunological and targeted therapeutic value, providing a new approach to GC treatment. [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]

Objectives: Cell surface glycosylation can influence protein-protein interactions with particular relevance to changes in core fucosylation and terminal sialylation. Glycans are ligands for immune regulatory lectin families like galectins (Gals) or sialic acid immunoglobulin-like lectins (Siglecs). This study delves into the glycan alterations within immune subsets of systemic lupus erythematosus (SLE). Methods: Evaluation of binding affinities of Galectin-1, Galectin-3, Siglec-1, Aleuria aurantia lectin (AAL, recognizing core fucosylation), and Sambucus nigra agglutinin (SNA, specific for α-2,6-sialylation) was conducted on various immune subsets in peripheral blood mononuclear cells (PBMCs) from control and SLE subjects. Lectin binding was measured by multi-parameter flow cytometry in 18 manually gated subsets of T-cells, NK-cells, NKT-cells, B-cells, and monocytes in unstimulated resting state and also after 3-day activation. Stimulated pre-gated populations were subsequently clustered by FlowSOM algorithm based on lectin binding and activation markers, CD25 or HLA-DR. Results: Elevated AAL, SNA and CD25+/CD25- SNA binding ratio in certain stimulated SLE T-cell subsets correlated with SLE Disease Activity Index 2000 (SLEDAI-2K) scores. The significantly increased frequencies of activated AALlow Siglec-1low NK metaclusters in SLE also correlated with SLEDAI-2K indices. In SLE, activated double negative NKTs displayed significantly lower core fucosylation and CD25+/CD25- Siglec-1 binding ratio, negatively correlating with disease activity. The significantly enhanced AAL binding in resting SLE plasmablasts positively correlated with SLEDAI-2K scores. Conclusion: Alterations in the glycosylation of immune cells in SLE correlate with disease severity, which might represent potential implications in the pathogenesis of SLE. [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]

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]

Aberrant glycosylation is a crucial strategy employed by cancer cells to evade cellular immunity. However, it's unclear whether homologous recombination (HR) status-dependent glycosylation can be therapeutically explored. Here, we show that the inhibition of branched N-glycans sensitizes HR-proficient, but not HR-deficient, epithelial ovarian cancers (EOCs) to immune checkpoint blockade (ICB). In contrast to fucosylation whose inhibition sensitizes EOCs to anti-PD-L1 immunotherapy regardless of HR-status, we observe an enrichment of branched N-glycans on HR-proficient compared to HR-deficient EOCs. Mechanistically, BRCA1/2 transcriptionally promotes the expression of MGAT5, the enzyme responsible for catalyzing branched N-glycans. The branched N-glycans on HR-proficient tumors augment their resistance to anti-PD-L1 by enhancing its binding with PD-1 on CD8+ T cells. In orthotopic, syngeneic EOC models in female mice, inhibiting branched N-glycans using 2-Deoxy-D-glucose sensitizes HR-proficient, but not HR-deficient EOCs, to anti-PD-L1. These findings indicate branched N-glycans as promising therapeutic targets whose inhibition sensitizes HR-proficient EOCs to ICB by overcoming immune evasion. Cancer cells can employ aberrant glycosylation patterns to evade the host immune response. Here the authors report that inhibition of branched N-glycans sensitizes homologous recombination (HR)-proficient, but not HR-deficient, epithelial ovarian cancer to immune checkpoint inhibitors. [ABSTRACT FROM AUTHOR]

Background: Hepatocellular carcinoma (HCC) ranks as the third most common cause of cancer related death globally, representing a substantial challenge to global healthcare systems. In China, the primary risk factor for HCC is the hepatitis B virus (HBV). Aberrant serum glycoconjugate levels have long been linked to the progression of HBV-associated HCC (HBV-HCC). Nevertheless, few study systematically explored the dysregulation of glycoconjugates in the progression of HBV-associated HCC and their potency as the diagnostic and prognostic biomarker. Methods: An integrated strategy that combined transcriptomics, glycomics, and glycoproteomics was employed to comprehensively investigate the dynamic alterations in glyco-genes, N-glycans, and glycoproteins in the progression of HBV- HCC. Results: Bioinformatic analysis of Gene Expression Omnibus (GEO) datasets uncovered dysregulation of fucosyltransferases (FUTs) in liver tissues from HCC patients compared to adjacent tissues. Glycomic analysis indicated an elevated level of fucosylated N-glycans, especially a progressive increase in fucosylation levels on IgA1 and IgG2 determined by glycoproteomic analysis. Conclusions: The findings indicate that the abnormal fucosylation plays a pivotal role in the progression of HBV-HCC. Systematic and integrative multi-omic analysis is anticipated to facilitate the discovery of aberrant glycoconjugates in tumor progression. [ABSTRACT FROM AUTHOR]

The RTS,S/AS02A malaria vaccine is based on the Plasmodium falciparum circumsporozoite protein (PfCSP), which is O -fucosylated on the sporozoite surface. We determined whether RTS,S/AS02A-induced immunoglobulin G (IgG) antibodies recognize vaccine-like nonfucosylated PfCSP better than native-like fucosylated PfCSP. Similar to previous vaccine trials, RTS,S/AS02A vaccination induced high anti-PfCSP IgG levels associated with malaria protection. IgG recognition of nonfucosylated and fucosylated PfCSP was equivalent, suggesting that PfCSP fucosylation does not affect antibody recognition. Clinical Trials Registration. NCT00197041. [ABSTRACT FROM AUTHOR]

CD14 is an innate immune receptor that senses pathogen-associated molecular patterns, such as lipopolysaccharide, to activate the innate immune response. Although CD14 is known to be glycosylated, detailed understanding about the structural and functional significance of this modification is still missing. Herein, an NMR and MS-based study, assisted by MD simulations, has provided a 3D-structural model of glycosylated CD14. Our results reveal the existence of a key N-glycosylation site at Asn282 that exclusively contains unprocessed oligomannnose N-glycans that perfectly fit the concave cavity of the bent-solenoid shaped protein. This site is not accessible to glycosidases and is fundamental for protein folding and secretion. A second N-site at Asn151 displays mostly complex N-glycans, with the typical terminal epitopes of the host cell-line expression system (i.e. βGal, α2,3 and α2,6 sialylated βGal, here), but also particularities, such as the lack of core fucosylation. The glycan at this site points outside the protein surface, resulting in N-glycoforms fully exposed and available for interactions with lectins. In fact, NMR experiments show that galectin-4, proposed as a binder of CD14 on monocytes to induce their differentiation into macrophages-like cells, interacts in vitro with CD14 through the recognition of the terminal glycoepitopes on Asn151. This work provides key information about CD14 glycosylation, which helps to better understand its functional roles and significance. Although protein glycosylation is known to be dynamic and influenced by many factors, some of the features found herein (presence of unprocessed N-glycans and lack of core Fuc) are likely to be protein specific. [ABSTRACT FROM AUTHOR]

Copyright of Science & Technology of Food Industry is the property of Science & Technology of Food Industry Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

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]

FUT8, the sole glycosyltransferase responsible for N-glycan core fucosylation, plays a crucial role in tumorigenesis and development. Aberrant FUT8 expression disrupts the function of critical cellular components and triggers the abnormality of tumor signaling pathways, leading to malignant transformations such as proliferation, invasion, metastasis, and immunosuppression. The association between FUT8 and unfavorable outcomes in various tumors underscores its potential as a valuable diagnostic marker. Given the remarkable variation in biological functions and regulatory mechanisms of FUT8 across different tumor types, gaining a comprehensive understanding of its complexity is imperative. Here, we review how FUT8 plays roles in tumorigenesis and development, and how this outcome could be utilized to develop potential clinical therapies for tumors. [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]

Lung cancer remains a global health crisis, responsible for significant morbidity and mortality. Late-stage diagnosis often limits treatment options and patient survival. Therefore, identifying reliable and sensitive biomarkers for early detection is crucial. Glycosylation, the addition of glycans to protein/RNA/lipid, is a vital cellular process. Normal glycosylation regulates healthy cell function, while alterations, particularly in fucosylation and sialylation, contribute to lung cancer development and progression. These aberrant glycosylation patterns are associated with processes such as immune modulation, cell migration, proliferation, and cell-cell recognition. Fucosylation, a specific type of glycosylation, is frequently altered in lung cancer, with high levels detected in tumors. Understanding the mechanisms behind this altered fucosylation holds immense potential. It can pave the way for the development of novel therapeutic and diagnostic tools for lung cancer. By analyzing specific fucosylation patterns in bodily fluids, it could lead to early-stage diagnosis. This review delves into the mechanisms of fucosylation in lung cancer initiation and metastasis, proposing promising strategies to target the mechanisms, aiming to inhibit tumor growth and disease progression. [ABSTRACT FROM AUTHOR]

Inflammatory bowel disease (IBD) is a common immune-mediated condition with its molecular pathogenesis remaining to be fully elucidated. This study aimed to deepen our understanding of the role of FUT2 in human IBD, by studying a new surrogate gene Sec1, a neighboring gene of Fut2 and Fut1 that co-encodes the α 1,2 fucosyltransferase in mice. CRISPR/Cas9 was used to prepare Sec1 knockout (Sec1−/−) mice. IBD was induced in mice using 3% w/v dextran sulphate sodium. Small interfering RNA (siRNA) was employed to silence Sec1 in murine colon cancer cell lines CT26.WT and CMT93. IBD-related symptoms, colonic immune responses, proliferation and apoptosis of colon epithelial cells were assessed respectively to determine the role of Sec1 in mouse IBD. Impact of Sec1 on the expression of death receptor 5 (DR5) and other apoptosis-associated proteins were determined. Sec1 knockout was found to be associated with deterioration of IBD in mice and elevated immune responses in the colonic mucosa. Silencing Sec1 in CT26.WT and CMT93 cells led to greater secretion of inflammatory cytokines IL-1β, IL-6 and TNF-α. Cell counting kit 8 (CCK8) assay, flow cytometry and TUNEL detection suggested that Sec1 expression promoted the proliferation of colon epithelial cells, inhibited cell apoptosis, reduced cell arrest in G0/G1 phase and facilitated repair of inflammatory injury. Over-expression of DR5 and several apoptosis-related effector proteins was noticed in Sec1−/− mice and Sec1-silenced CT26.WT and CMT93 cells, supporting a suppressive role of Sec1 in cell apoptosis. Our results depicted important regulatory roles of Sec1 in mouse IBD, further reflecting the importance of FUT2 in the pathogenesis of human IBD. Research highlights: Sec1, a proxy gene of human fut2, plays a protective role against mouse IBD. Sec1 negatively modulates the secretion of key intestinal inflammatory factors. Sec1 supports cell proliferation and regulates DR5 fucosylation and IEC apoptosis. [ABSTRACT FROM AUTHOR]

Aberrant glycosylation is a hallmark of cancer and is not just a consequence, but also a driver of a malignant phenotype. In prostate cancer, changes in fucosylated and sialylated glycans are common and this has important implications for tumor progression, metastasis, and immune evasion. Glycans hold huge translational potential and new therapies targeting tumor-associated glycans are currently being tested in clinical trials for several tumor types. Inhibitors targeting fucosylation and sialylation have been developed and show promise for cancer treatment, but translational development is hampered by safety issues related to systemic adverse effects. Recently, potent metabolic inhibitors of sialylation and fucosylation were designed that reach higher effective concentrations within the cell, thereby rendering them useful tools to study sialylation and fucosylation as potential candidates for therapeutic testing. Here, we investigated the effects of global metabolic inhibitors of fucosylation and sialylation in the context of prostate cancer progression. We find that these inhibitors effectively shut down the synthesis of sialylated and fucosylated glycans to remodel the prostate cancer glycome with only minor apparent side effects on other glycan types. Our results demonstrate that treatment with inhibitors targeting fucosylation or sialylation decreases prostate cancer cell growth and downregulates the expression of genes and proteins important in the trajectory of disease progression. We anticipate our findings will lead to the broader use of metabolic inhibitors to explore the role of fucosylated and sialylated glycans in prostate tumor pathology and may pave the way for the development of new therapies for prostate cancer. [ABSTRACT FROM AUTHOR]

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) can occur due to maternal IgG antibodies targeting platelet antigens, causing life-threatening bleeding in the neonate. However, the disease manifests itself in only a fraction of pregnancies, most commonly with anti-HPA-1a antibodies. We found that in particular, the core fucosylation in the IgG-Fc tail is highly variable in anti-HPA-1a IgG, which strongly influences the binding to leukocyte IgG-Fc receptors IIIa/b (FcγRIIIa/b). Currently, gold-standard IgG-glycoanalytics rely on complicated methods (e.g., mass spectrometry (MS)) that are not suited for diagnostic purposes. Our aim was to provide a simplified method to quantify the biological activity of IgG antibodies targeting cells. We developed a cellular surface plasmon resonance imaging (cSPRi) technique based on FcγRIII-binding to IgG-opsonized cells and compared the results with MS. The strength of platelet binding to FcγR was monitored under flow using both WT FcγRIIIa (sensitive to Fc glycosylation status) and mutant FcγRIIIa-N162A (insensitive to Fc glycosylation status). The quality of the anti-HPA-1a glycosylation was monitored as the ratio of binding signals from the WT versus FcγRIIIa-N162A, using glycoengineered recombinant anti-platelet HPA-1a as a standard. The method was validated with 143 plasma samples with anti-HPA-1a antibodies analyzed by MS with known clinical outcomes and tested for validation of the method. The ratio of patient signal from the WT versus FcγRIIIa-N162A correlated with the fucosylation of the HPA-1a antibodies measured by MS (r=-0.52). Significantly, FNAIT disease severity based on Buchanan bleeding score was similarly discriminated against by MS and cSPRi. In conclusion, the use of IgG receptors, in this case, FcγRIIIa, on SPR chips can yield quantitative and qualitative information on platelet-bound anti-HPA-1a antibodies. Using opsonized cells in this manner circumvents the need for purification of specific antibodies and laborious MS analysis to obtain qualitative antibody traits such as IgG fucosylation, for which no clinical test is currently available. [ABSTRACT FROM AUTHOR]

Introduction: Immunoglobulin G (IgG) contains a conserved N-glycan in the fragment crystallizable (Fc), modulating its structure and effector functions. In anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) alterations of IgG Fc-glycosylation have been observed to correlate with the disease course. Here, we examined longitudinal changes in N-linked Fc glycans of IgG in an AAV patient cohort and their relationship with disease flares. Methods: Using liquid chromatography coupled with mass spectrometry, we analysed IgG Fc-glycosylation in 410 longitudinal samples from 96 individuals with AAV. Results: Analysis of the cross-sectional differences as well as longitudinal changes demonstrated that IgGs of relapsing PR3-ANCA patients have higher Fc-bisection at diagnosis (P= 0.004) and exhibit a decrease in Fc-sialylation prior to the relapse (P= 0.0004), discriminating them from non-relapsing patients. Most importantly, PR3-ANCA patients who experienced an ANCA rise and relapsed shortly thereafter, exhibit lower IgG Fc-fucosylation levels compared to non-relapsing patients already 9 months before relapse (P= 0.02). Discussion: Our data indicate that IgG Fc-bisection correlates with long-term treatment outcome, while lower IgG Fc-fucosylation and sialylation associate with impending relapse. Overall, our study replicated the previously published reduction in total IgG Fc-sialylation at the time of relapse, but showed additionally that its onset precedes relapse. Furthermore, our findings on IgG fucosylation and bisection are entirely new. All these IgG Fc-glycosylation features may have the potential to predict a relapse either independently or in combination with known risk factors, such as a rise in ANCA titre. [ABSTRACT FROM AUTHOR]

Prior research on cholera toxin (CT) binding and intoxication has relied on human colonic cancer derived epithelial cells. While these transformed cell lines have been beneficial, they neither derive from small intestine where intoxication occurs, nor represent the diversity of small intestinal epithelial cells (SI-ECs) and variation in glycoconjugate expression among individuals. Here, we used human enteroids, derived from jejunal biopsies of multipledonors to study CT binding and intoxication of human non-transformed SI-ECs. We modulated surface expression of glycosphingolipids, glycoproteins and specific glycans to distinguish the role of each glycan/glycoconjugate. Cholera-toxin-subunit-B (CTB) mutants were generated to decipher the preference of each glycoconjugate to different binding sites and the correlation between CT binding and intoxication. Human enteroids contain trace amounts of GM1, but other glycosphingolipids may be contributing to CT intoxication. We discovered that inhibition of either fucosylation or O-glycosylation sensitize enteroids to CT-intoxication. This can either be a consequence of the removal of fucosylated "decoy-like-ligands" binding to CTB's non-canonical site and/or increase in the availability of Gal/GalNAc-terminating glycoconjugates binding to the canonical site. Furthermore, simultaneous inhibition of fucosylation and O-glycosylation increased the availability of additional Gal/GalNAc-terminating glycoconjugates but counteracted the sensitization in CT intoxication caused by inhibiting O-glycosylation because of reduction in fucose. This implies a dual role of fucose as a functional glycan and a decoy, the interplay of which influences CT binding and intoxication. Finally, while the results were similar for enteroids from different donors, they were not identical, pointing to a role for human genetic variation in determining sensitivity to CT. [ABSTRACT FROM AUTHOR]

Neutrophils store microbicidal glycoproteins in cytosolic granules to fight intruding pathogens, but their granule distribution and formation mechanism(s) during granulopoiesis remain unmapped. Herein, we comprehensively profile the neutrophil N-glycoproteome with spatiotemporal resolution by analyzing four key types of intracellular organelles isolated from blood-derived neutrophils and during their maturation from bone marrow–derived progenitors using a glycomics-guided glycoproteomics approach. Interestingly, the organelles of resting neutrophils exhibited distinctive glycophenotypes including, most strikingly, highly truncated N-glycans low in α2,6-sialylation and Lewis fucosylation decorating a diverse set of microbicidal proteins (e.g., myeloperoxidase, azurocidin, neutrophil elastase) in the azurophilic granules. Excitingly, proteomics and transcriptomics data from discrete myeloid progenitor stages revealed that profound glycoproteome remodeling underpins the promyelocytic-to-metamyelocyte transition and that the glycophenotypic differences are driven primarily by dynamic changes in protein expression and less by changes within the glycosylation machinery. Notable exceptions were the oligosaccharyltransferase subunits responsible for initiation of N-glycoprotein biosynthesis that were strongly expressed in early myeloid progenitors correlating with relatively high levels of glycosylation of the microbicidal proteins in the azurophilic granules. Our study provides spatiotemporal insights into the complex neutrophil N-glycoproteome featuring intriguing organelle-specific N-glycosylation patterns formed by dynamic glycoproteome remodeling during the early maturation stages of the myeloid progenitors. [ABSTRACT FROM AUTHOR]

Fucose is a deoxyhexose sugar present and studied in mammals. The process of fucosylation has been the primary focus in studies relating to fucose in animals due to the presence of fucose in Lewis antigens. Very few studies have reported its presence in Fungi, mostly in Mucoromycotina. The constitution of 25% and 12% of this sugar in the carbohydrates of cell wall in the respective Umbelopsis and Mucorales strains boosts the need to bridge the gap of knowledge on fucose metabolism across the fungal tree of life. In the absence of a network map involving fucose proteins, we carried out an in-silico approach to construct the fucose metabolic map in Fungi. We analyzed the taxonomic distribution of 85 protein families in Fungi including diverse early diverging fungal lineages. The expression of fucose-related protein-coding genes proteins was validated with the help of transcriptomic data originating from representatives of early diverging fungi. We found proteins involved in several metabolic activities apart from fucosylation such as synthesis, transport and binding. Most of the identified protein families are shared with Metazoa suggesting an ancestral origin in Opisthokonta. However, the overall complexity of fucose metabolism is greater in Metazoa than in Fungi. Massive gene loss has shaped the evolutionary history of these metabolic pathways, leading to a repeated reduction of these pathways in most yeast-forming lineages. Our results point to a distinctive mode of utilization of fucose among fungi belonging to Dikarya and the early diverging lineages. We speculate that, while Dikarya used fucose as a source of nutrients for metabolism, the early diverging group of fungi depended on fucose as a building block and signaling compound. [ABSTRACT FROM AUTHOR]

Pulmonary arterial hypertension (PAH) is a progressive cardiopulmonary disease with unclear underlying molecular mechanisms and limited therapeutic options. This study aimed to explore the role of core fucosylation and the only glycosyltransferase FUT8 in PAH. We observed increased core fucosylation in a monocrotaline (MCT)-induced PAH rat model and isolated rat pulmonary artery smooth muscle cells (PASMCs) treated with platelet-derived growth factor-BB (PDGF-BB). We found that 2-fluorofucose (2FF), a drug used to inhibit core fucosylation, improved hemodynamics and pulmonary vascular remodeling in MCT-induced PAH rats. In vitro, 2FF effectively restrains the proliferation, migration, and phenotypic switching of PASMCs and promotes apoptosis. Compared with controls, serum FUT8 concentration in PAH patients and MCT-induced rats was significantly elevated. FUT8 expression appeared increased in the lung tissues of PAH rats, and the colocalization of FUT8 with a-SMA was also observed. SiRNA was used to knockdown FUT8 in PASMCs (siFUT8). After effectively silencing FUT8 expression, phenotypic changes induced in PASMCs by PDGF-BB stimulation were alleviated. FUT8 activated the AKT pathway, while the admission of AKT activator SC79 could partially counteract the negative effect of siFUT8 on the proliferation, apoptotic resistance, and phenotypic switching of PASMCs, which may be involved in the core fucosylation of vascular endothelial growth factor receptor (VEGFR). Our research confirmed the critical role of FUT8 and its mediated core fucosylation in pulmonary vascular remodeling in PAH, providing a potential novel therapeutic target for PAH. [ABSTRACT FROM AUTHOR]

Fucosylation, or the attachment of a fucose moiety to a glycan or protein by the action of fucosyltransferases, happens extensively in all living organisms. It plays a vital role in multiple biological processes from development to immunity, and is thought to be highly associated with the occurrence of many human diseases. While the general principles of fucosylation are similar in all organisms, most insects synthesize less processed fucosylated glycans compared to humans. Recent studies in insects show that disruption of fucosylation causes developmental defects leading to lethality, suggesting an essential role in insects. However, because of the limited information available, the molecular mechanisms behind these phenotypes remain unresolved. This review provides an overview on insect fucosylation, including the principle and function of fucosylation, the phylogenesis of the fucosyltransferases, and the diversity and abundance of fucosylated glycans. To provide a better understanding of the different roles of fucosylation in insects, knowledge on fucosylation in mammals or other invertebrates is discussed. As the dynamic requirement for fucosylation in insects needs more research to elucidate the underlying mechanisms, we hope this overview of fucosylation could provide new insights into its role in insects for future studies. [ABSTRACT FROM AUTHOR]

Antibody-dependent cell-mediated cytotoxicity (ADCC) by natural killer (NK) lymphocytes eliminates cells infected with viruses. Anti-viral ADCC requires three components: (1) antibody; (2) effector lymphocytes with the Fc-IgG receptor CD16A; and (3) viral proteins in infected cell membranes. Fc-afucosylated antibodies bind with greater affinity to CD16A than fucosylated antibodies; individuals' variation in afucosylation contributes to differences in ADCC. Current assays for afucosylated antibodies involve expensive methods. We report an improved bioassay for antibodies that supports ADCC, which encompasses afucosylation. This assay utilizes the externalization of CD107a by NK-92-CD16A cells after antibody recognition. We used anti-CD20 monoclonal antibodies, GA101 WT or glycoengineered (GE), 10% or ~50% afucosylated, and CD20-positive Raji target cells. CD107a increased detection 7-fold compared to flow cytometry to detect Raji-bound antibodies. WT and GE antibody effective concentrations (EC50s) for CD107a externalization differed by 20-fold, with afucosylated GA101-GE more detectable. The EC50s for CD107a externalization vs. 51Cr cell death were similar for NK-92-CD16A and blood NK cells. Notably, the % CD107a-positive cells were negatively correlated with dead Raji cells and were nearly undetectable at high NK:Raji ratios required for cytotoxicity. This bioassay is very sensitive and adaptable to assess anti-viral antibodies but unsuitable as a surrogate assay to monitor cell death after ADCC. [ABSTRACT FROM AUTHOR]

Epithelial cells can undergo apoptosis by manipulating the balance between pro-survival and apoptotic signals. In this work, we show that TRAIL-induced apoptosis can be differentially regulated by the expression of α(1,6)fucosyltransferase (FucT-8), the only enzyme in mammals that transfers the α(1,6)fucose residue to the pentasaccharide core of complex N-glycans. Specifically, in the cellular model of colorectal cancer (CRC) progression formed using the human syngeneic lines SW480 and SW620, knockdown of the FucT-8-encoding FUT8 gene significantly enhanced TRAIL-induced apoptosis in SW480 cells. However, FUT8 repression did not affect SW620 cells, which suggests that core fucosylation differentiates TRAIL-sensitive premetastatic SW480 cells from TRAIL-resistant metastatic SW620 cells. In this regard, we provide evidence that phosphorylation of ERK1/2 kinases can dynamically regulate TRAIL-dependent apoptosis and that core fucosylation can control the ERK/MAPK pro-survival pathway in which SW480 and SW620 cells participate. Moreover, the depletion of core fucosylation sensitises primary tumour SW480 cells to the combination of TRAIL and low doses of 5-FU, oxaliplatin, irinotecan, or mitomycin C. In contrast, a combination of TRAIL and oxaliplatin, irinotecan, or bevacizumab reinforces resistance of FUT8-knockdown metastatic SW620 cells to apoptosis. Consequently, FucT-8 could be a plausible target for increasing apoptosis and drug response in early CRC. [ABSTRACT FROM AUTHOR]

Background: The pathological mechanisms of SARS-CoV-2 in humans remain unclear and the unpredictability of COVID-19 progression may be attributed to the absence of biomarkers that contribute to the prognosis of this disease. Therefore, the discovery of biomarkers is needed for reliable risk stratification and to identify patients who are more likely to progress to a critical stage. Methods: Aiming to identify new biomarkers we analysed N-glycan traits in plasma from 196 patients with COVID-19. Samples were classified into three groups according to their severity (mild, severe and critical) and obtained at diagnosis (baseline) and at 4 weeks of follow-up (postdiagnosis), to evaluate their behaviour through disease progression. N-glycans were released with PNGase F and labelled with Rapifluor-MS, followed by their analysis by LC-MS/MS. The Simglycan structural identification tool and Glycostore database were employed to predict the structure of glycans. Results: We determined that plasma from SARS-CoV-2-infected patients display different N-glycosylation profiles depending on the disease severity. Specifically, levels of fucosylation and galactosylation decreased with increasing severity and Fuc1Hex5HexNAc5 was identified as the most suitable biomarker to stratify patients at diagnosis and distinguish mild from critical outcomes. Conclusion: In this study we explored the global plasma glycosignature, reflecting the inflammatory state of the organs during the infectious disease. Our findings show the promising potential of glycans as biomarkers of COVID-19 severity. [ABSTRACT FROM AUTHOR]

Mac-2 binding protein (Mac-2bp) is a serum glycoprotein that contains seven N- glycans, and Mac-2bp serum levels are increased in patients with several types of cancer and liver disease. Mac-2bp glycosylation isomer has been applied as a clinical biomarker of several diseases, including liver fibrosis. In the present study, we identified fucosylated Mac-2bp in the conditioned medium of cancer cells resistant to anticancer therapies using glycoproteomic analyses. Fucosylation is one of the most important types of glycosylation involved in carcinogenesis and cancer stemness. To establish a next-generation glycan antibody for fucosylated Mac-2bp, we used fucosylation-deficient HEK293T cells to prepare reference Mac-2bp antigens and performed antibody screening. Unexpectedly, the 19-8H mAb obtained with our screen recognized 70K Mac-2bp, which is C-terminus-truncated product, rather than specifically recognizing fucosylated Mac-2bp. We performed immunocytochemistry using our novel 19-8H mAb, which resulted in strong cell surface staining of anticancer drug-resistant cancer cells. Therefore, our novel 19-8H mAb represents a valuable tool for cancer biology research that can help elucidate the biological function of 70K Mac-2bp. [ABSTRACT FROM AUTHOR]

Changes in protein glycosylation are associated with most biological processes, and the importance of glycomic analysis in the research of disorders is constantly increasing, including in the neurodevelopmental field. We glycoprofiled sera in 10 children with attention-deficit hyperactivity disorder (ADHD) and 10 matching healthy controls for 3 types of samples: whole serum, sera after depletion of abundant proteins (albumin and IgG), and isolated IgG. The analytical methods used were a lectin-based glycoprotein microarray enabling high-throughput glycan analysis and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) as a standard method for the identification of glycan structures. For microarray analysis, the samples printed on microarray slides were incubated with biotinylated lectins and detected using the fluorescent conjugate of streptavidin by a microarray scanner. In the ADHD patient samples, we found increased antennary fucosylation, decreased di-/triantennary N-glycans with bisecting N-acetylglucosamine (GlcNAc), and decreased α2-3 sialylation. The results obtained by both independent methods were consistent. The study's sample size and design do not allow far-reaching conclusions to be drawn. In any case, there is a strong demand for a better and more comprehensive diagnosis of ADHD, and the obtained results emphasize that the presented approach brings new horizons to studying functional associations of glycan alterations in ADHD. [ABSTRACT FROM AUTHOR]

Background: Cholangiocarcinoma (CCA) is a silent tumor with a high mortality rate due to the difficulty of early diagnosis and prediction of recurrence even after timely surgery. Serologic cancer biomarkers have been used in clinical practice, but their low specificity and sensitivity have been problematic. In this study, we aimed to identify CCA-specific glycan epitopes that can be used for diagnosis and to elucidate the mechanisms by which glycosylation is altered with tumor progression. Methods: The serum of patients with various cancers was fractioned into membrane-bound and soluble components using serial ultracentrifugation. Lectin blotting was conducted to evaluate glycosylation. Proteins having altered glycosylation were identified using proteomic analysis and further confirmed using immunoblotting analysis. We performed HPLC, gene analysis, real-time cargo tracking, and immunohistochemistry to determine the origin of CCA glycosylation and its underlying mechanisms. Extracellular vesicles (EV) were isolated from the sera of 62 patients with CCA at different clinical stages and inflammatory conditions and used for glycan analysis to assess their clinical significance. Results: The results reveal that glycosylation patterns between soluble and membrane-bound fractions differ significantly even when obtained from the same donor. Notably, glycans with a1-3/4 fucose and b1-6GlcNAc branched structures increase specifically in membrane-bound fractions of CCA. Mechanically, it is primarily due to b-haptoglobin (b-Hp) originating from CCA expressing fucosyltransferase-3/4 (FUT 3/4) and N-acetylglucosaminyltransferase-V (MGAT5). Newly synthesized b-Hp is loaded into EVs in early endosomes via a KFERQ-like motif and then secreted from CCA cells to induce tumor progression. In contrast, b-Hp expressed by hepatocytes is secreted in a soluble form that does not affect CCA progression. Moreover, evaluation of EV glycosylation in CCA patients shows that fucosylation level of EV-Hp gradually increases with tumor progression and decreases markedly when the tumors are eliminated by surgery. Conclusion: This study suggests that terminal fucosylation of Hp in cancerderived exosomes can be a novel glycan marker for diagnosis and prognosis of CCA. These findings highlight the potential of glycan analysis in different fractions of serum for biomarker discover for other diseases. Further research is needed to understand the role of fucosylated EVs on CCA progression. [ABSTRACT FROM AUTHOR]

Glycobiology as a field holds enormous potential for understanding human health and disease. However, few glycobiology studies adequately address the issue of sex differences in biology, which severely limits the conclusions that can be drawn. Numerous CAZymes, lectins, and other carbohydrate-associated molecules have the potential to be differentially expressed and regulated with sex, leading to differences in O-GlcNAc, N-glycan branching, fucosylation, sialylation, and proteoglycan structure, among others. Expression of proteins involved in glycosylation is influenced through hormones, miRNA, and gene dosage effects. In this review, we discuss the benefits of incorporating sex-based analysis in glycobiology research and the potential drivers of sex differences. We highlight examples of where incorporation of sex-based analysis has led to insights into glycobiology. Finally, we offer suggestions for how to proceed moving forward, even if the experiments are already complete. Properly incorporating sex based analyses into projects will substantially improve the accuracy and reproducibility of studies as well as accelerate the rate of discovery in the glycosciences. [ABSTRACT FROM AUTHOR]

The Arabidopsis sensitive-to-freezing8 (sfr8) mutant exhibits reduced cell wall (CW) fucose levels and compromised freezing tolerance. To examine whether CW fucosylation also affects the response to desiccation, we tested the effect of leaf excision in sfr8 and the allelic mutant mur1-1. Leaf water loss was strikingly higher than in the wild type in these, but not other, fucosylation mutants. We hypothesized that reduced fucosylation in guard cell (GC) walls might limit stomatal closure through altering mechanical properties. Multifrequency atomic force microscopy (AFM) measurements revealed a reduced elastic modulus (E ʹ), representing reduced stiffness, in sfr8 GC walls. Interestingly, however, we discovered a compensatory mechanism whereby a concomitant reduction in the storage modulus (E ʹʹ) maintained a wild-type viscoelastic time response (tau) in sfr8. Stomata in intact leaf discs of sfr8 responded normally to a closure stimulus, abscisic acid, suggesting that the time response may relate more to closure properties than stiffness does. sfr8 stomatal pore complexes were larger than those of the wild type, and GCs lacked a fully developed cuticular ledge, both potential contributors to the greater leaf water loss in sfr8. We present data that indicate that fucosylation-dependent dimerization of the CW pectic domain rhamnogalacturonan-II may be essential for normal cuticular ledge development and leaf water retention. [ABSTRACT FROM AUTHOR]

Fucosylated proteins are widely used as biomarkers of cancer and inflammation. Fucosylated alpha-fetoprotein (AFP-L3) is a specific biomarker for hepatocellular carcinoma. We previously showed that increases in serum AFP-L3 levels depend on increased expression of fucosylation-regulatory genes and abnormal transport of fucosylated proteins in cancer cells. In normal hepatocytes, fucosylated proteins are selectively secreted in the bile duct but not blood. In cases of cancer cells without cellular polarity, this selective secretion system is destroyed. Here, we aimed to identify cargo proteins involved in the selective secretion of fucosylated proteins, such as AFP-L3, into bile duct-like structures in HepG2 hepatoma cells, which have cellular polarity like, in part, normal hepatocytes. α1-6 Fucosyltransferase (FUT8) is a key enzyme to synthesize core fucose and produce AFP-L3. Firstly, we knocked out the FUT8 gene in HepG2 cells and investigated the effects on the secretion of AFP-L3. AFP-L3 accumulated in bile duct-like structures in HepG2 cells, and this phenomenon was diminished by FUT8 knockout, suggesting that HepG2 cells have cargo proteins for AFP-L3. To identify cargo proteins involved in the secretion of fucosylated proteins in HepG2 cells, immunoprecipitation and the proteomic Strep-tag system experiments followed by mass spectrometry analyses were performed. As a result of proteomic analysis, seven kinds of lectin-like molecules were identified, and we selected vesicular integral membrane protein gene VIP36 as a candidate of the cargo protein that interacts with the α1-6 fucosylation (core fucose) on N-glycan according to bibliographical consideration. Expectedly, the knockout of the VIP36 gene in HepG2 cells suppressed the secretion of AFP-L3 and other fucosylated proteins, such as fucosylated alpha-1 antitrypsin, into bile duct-like structures. We propose that VIP36 could be a cargo protein involved in the apical secretion of fucosylated proteins in HepG2 cells. [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]

Background: Fucosyltransferase 2(FUT2) and its induced α-1,2 fucosylation is associated with cancer metastasis. However, the role of FUT2 in colorectal cancer (CRC) metastasis remains unclear. Methods: The expression levels and clinical analyses of FUT2 were assessed in CRC samples. Migration and invasion assays, EMT detection, nude mice peritoneal dissemination models and intestinal specific FUT2 knockout mice (FUT2△IEC mice) were used to investigate the effect of FUT2 on metastasis in colorectal cancer. Quantitative proteomics study of glycosylated protein, UEA enrichment, Co-immunoprecipitation identified the mediator of the invasive-inhibiting effects of FUT2. Results: FUT2 is downregulated in CRC tissues and is positively correlated with the survival of CRC patients. FUT2 is an inhibitor of colorectal cancer metastasis which, when overexpressed, suppresses invasion and tumor dissemination in vitro and in vivo. FUT2 knock-out mice (FUT2△IEC mice) develop AMO and DSS-induced tumors and promote EMT in colorectal cancers. FUT2-induced α-1,2 fucosylation impacts the ability of low-density lipoprotein receptor-related protein 1(LRP1) to suppress colorectal cancer invasion. Conclusions: Our study demonstrated that FUT2 induces α-1,2 fucosylation and inhibits EMT and metastasis of colorectal cancer through LRP1 fucosylation, suggesting that FUT2 may serve as a therapeutic target for colorectal cancer. 8zeohEanYa54_QDWABytw7 Video Abstract [ABSTRACT FROM AUTHOR]

Various tumors rely on post-translational modifications (PTMs) to promote invasiveness and angiogenesis and to reprogram cellular energetics to abate anti-cancer immunity. Among PTMs, fucosylation is a particular type of glycosylation that has been linked to different aspects of immune and hormonal physiological functions as well as hijacked by many types of tumors. Multiple tumors, including breast cancer, have been linked to dismal prognoses and increased metastatic potential due to fucosylation of the glycan core, namely core-fucosylation. Pre-clinical studies have examined the molecular mechanisms regulating core-fucosylation in breast cancer models, its negative prognostic value across multiple disease stages, and the activity of in vivo pharmacological inhibition, instructing combinatorial therapies and translation into clinical practice. Throughout this review, we describe the role of fucosylation in solid tumors, with a particular focus on breast cancer, as well as physiologic conditions on the immune system and hormones, providing a view into its potential as a biomarker for predicating or predicting cancer outcomes, as well as a potential clinical actionability as a biomarker. [ABSTRACT FROM AUTHOR]