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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]

[Display omitted] • This study first reveals a significant presence of core fucosylation in female fertility control. • Ovarian response and oocyte quality are associated with core fucosylation. • Core fucosylation regulates FSH responsiveness of the FSHR on granulosa cells in follicles. • Core fucosylation levels among young and older women could be an indicator for monitoring reproductive health. • Appropriate dietary management of L-fucose appears to be considered as a potentially safe means to maintain and restore reproductive capacity. Ovarian low response to follicle-stimulating hormone (FSH) causes infertility featuring hypergonadotropic hypogonadism, ovarian failure, and/or defective ovarian response. N-glycosylation is essential for FSH receptor (FSHR). Core fucosylation catalyzed by fucosyltransferase 8 (FUT8) is the most common N-glycosylation. Core fucosylation level changes between individuals and plays important roles in multiple physiological and pathological conditions. This study aims to elucidate the significance of FUT8 to modulate FSHR function in female fertility. Samples from patients classified as poor ovary responders (PORs) were detected with lectin blot and real-time PCR. Fut8 gene knockout (Fut8−/−) mice and FUT8-knockdown human granulosa cell line (KGN-KD) were established and in vitro fertilization (IVF) assay, western blot, molecular interaction, immunofluorescence and immunoprecipitation were applied. Core fucosylation is indispensable for oocyte and follicular development. FSHR is a highly core-fucosylated glycoprotein. Loss of core fucosylation suppressed binding of FSHR to FSH, and attenuated FSHR downstream signaling in granulosa cells. Transcriptomic analysis revealed the downregulation of several transcripts crucial for oocyte meiotic progression and preimplantation development in Fut8−/− mice and in POR patients. Furthermore, loss of FUT8 inhibited the interaction between granulosa cells and oocytes, reduced transzonal projection (TZP) formation and caused poor developmental competence of oocytes after fertilization in vitro. While L-fucose administration increased the core fucosylation of FSHR, and its sensitivity to FSH. This study first reveals a significant presence of core fucosylation in female fertility control. Decreased fucosylation on FSHR reduces the interaction of FSH-FSHR and subsequent signaling, which is a feature of the POR patients. Our results suggest that core fucosylation controls oocyte and follicular development via the FSH/FSHR pathway and is essential for female fertility in mammals. [ABSTRACT FROM AUTHOR]

Culture conditions have a profound impact on therapeutic protein production and glycosylation, a critical therapeutic-quality attribute, especially for monoclonal antibodies (mAbs). While the critical culture parameter of pH has been known since the early 1990s to affect protein glycosylation and production, detailed glycan and metabolic characterization and mechanistic understanding are critically lacking. Here, Chinese Hamster Ovary (CHO) cells were grown in bioreactors at pH 6.75, 7, and 7.25 (± 0.03) to examine how pH affects cell metabolism and site-specific N-linked glycosylation of the produced broadly neutralizing anti-HIV IgG1 mAb. VRC01 has N-linked glycosylation sites in both the Fc region and the Fab region, a situation not previously examined with respect to mAb glycosylation as affected by culture conditions. Using parsimonious Flux Balance Analysis (pFBA) and Flux Variability Analysis (FVA), we dissect and quantitate the impact of pH on cell growth, glucose/lactate metabolism, accumulation of the toxic metabolite ammonia, IgG production rates, and nonessential amino acid metabolism. pFBA revealed that beyond the established mechanism of glutamine conversion to glutamate, ammonia is also produced by the reaction converting serine to pyruvate, especially in the later phases of culture. pFBA also provided insights into the switch from ammonia production to consumption, notably due to depletion of glutamine, and consumption of glutamate and aspartate. We document that culture duration and pH alter the complex bimodal patterns (production/uptake) of several essential and non-essential amino acids. Site-specific N-linked glycan analysis using glycopeptide mapping demonstrated that pH significantly affects the glycosylation profiles of the two IgG1 sites. Fc region glycans were completely fucosylated but did not contain any sialylation. The Fab region glycans were not completely fucosylated but contained sialylated glycans. Bioreactor pH affected both the fucosylation and sialylation indexes in the Fab region and the galactosylation index of the Fc region. However, fucosylation in the Fc region was unaffected thus demonstrating that the effect of pH on site-specific N-linked glycosylation is complex. • Culture pH impacts cell growth, mAb synthesis, glycolysis, amino acid metabolism, ammonia synthesis, and mAb glycosylation. • Parsimonious flux balance analysis illuminates the dynamic impact of bioreactor pH on cell metabolism and mAb formation. • Glycopeptide mapping revealed significant differences in Fab and Fc glycans of the IgG1 antibody. • Bioreactor pH impacted fucosylation and sialylation in the Fab region and galactosylation in the Fc region of the mAb. [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]

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]

Fucosylation, especially core fucosylation of N-glycans catalyzed by α1-6 fucosyltransferase (fucosyltransferase 8 or FUT8), plays an important role in regulating the peripheral immune system and inflammation. However, its role in microglial activation is poorly understood. Here we used human induced pluripotent stem cells-derived microglia (hiMG) as a model to study the role of FUT8-catalyzed core fucosylation in amyloid-β oligomer (AβO)-induced microglial activation, in view of its significant relevance to the pathogenesis of Alzheimer's disease (AD). HiMG responded to AβO and lipopolysaccharides (LPS) with a pattern of pro-inflammatory activation as well as enhanced core fucosylation and FUT8 expression within 24 h. Furthermore, we found increased FUT8 expression in both human AD brains and microglia isolated from 5xFAD mice, a model of AD-like cerebral amyloidosis. Inhibition of fucosylation in AβO-stimulated hiMG reduced the induction of pro-inflammatory cytokines, suppressed the activation of p38MAPK, and rectified phagocytic deficits. Specific inhibition of FUT8 by siRNA-mediated knockdown also reduced AβO-induced pro-inflammatory cytokines. We further showed that p53 binds to the two consensus binding sites in the Fut8 promoter, and that p53 knockdown abolished FUT8 overexpression in AβO-activated hiMG. Taken together, our evidence supports that FUT8-catalyzed core fucosylation is a signaling pathway required for AβO-induced microglia activation and that FUT8 is a component of the p53 signaling cascade regulating microglial behavior. Because microglia are a key driver of AD pathogenesis, our results suggest that microglial FUT8 could be an anti-inflammatory therapeutic target for AD.

B-type natriuretic peptide or N-terminal pro–B-type natriuretic peptide is the only blood biomarker in established risk calculators for pulmonary arterial hypertension (PAH). Profiling systemic-originated plasma immunoglobulin G (IgG) N-glycans, which reflect different components of the pathophysiology of PAH including immune dysregulation and inflammation, may improve PAH risk assessment. This study sought to identify plasma IgG N-glycan biomarkers that predict survival in PAH to improve risk assessment. This cohort study examined 622 PAH patients from 2 national centers (Beijing [discovery] cohort: n = 273; Shanghai [validation] cohort: n = 349). Plasma IgG N-glycomes were profiled by a robust mass spectrometry–based method. Prognostic IgG N-glycan traits were identified and validated in the 2 cohorts using Cox regression and Kaplan-Meier survival analyses. The added value of IgG N-glycan traits to previously established risk models was assessed using Harrell C-indexes and survival analysis. Plasma IgG fucosylation was found to predict survival independent of age and sex in the discovery cohort (HR: 0.377; 95% CI: 0.168-0.845; P = 0.018) with confirmation in the validation cohort (HR: 0.445; 95% CI: 0.264-0.751; P = 0.005). IgG fucosylation remained a robust predictor of mortality in combined cohorts after full adjustment and in subgroup analyses. Integrating IgG fucosylation into previously established risk models improved their predictive capacity, marked by an overall elevation in Harrell C-indexes. IgG fucosylation was useful in further stratifying the intermediate-risk patients classified by a previously established model. Plasma IgG fucosylation informs PAH prognosis independent of established factors, offering additional value for predicting PAH outcomes. [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]

a1,6-Fucosyltransferase (Fut8) is the enzyme responsible for catalyzing core fucosylation. Exogenous L-fucose upregulates fucosylation levels through the GDP-fucose salvage pathway. This study investigated the relationship between core fucosylation and immunoglobulin G (IgG) amounts in serum utilizing WT (Fut8+/+), Fut8 heterozygous knockout (Fut8+/- ), and Fut8 knockout (Fut8-/- ) mice. The IgG levels in serum were lower in Fut8+/- and Fut8-/- mice compared with Fut8+/+ mice. Exogenous L-fucose increased IgG levels in Fut8+/- mice, while the ratios of core fucosylated IgG versus total IgG showed no significant difference among Fut8+/+, Fut8+/-, and Fut8+/- mice treated with L-fucose. These ratios were determined by Western blot, lectin blot, and mass spectrometry analysis. Real-time PCR results demonstrated that mRNA levels of IgG Fc and neonatal Fc receptor, responsible for protecting IgG turnover, were similar among Fut8+/+, Fut8+/-, and Fut8+/- mice treated with L-fucose. In contrast, the expression levels of Fc-gamma receptor IV (FcgRIV), mainly expressed on macrophages and neutrophils, were increased in Fut8+/- mice compared to Fut8+/+ mice. The effect was reversed by administrating Lfucose, suggesting that core fucosylation primarily regulates the IgG levels through the Fc-FcgRIV degradation pathway. Consistently, IgG internalization and transcytosis were suppressed in FcgRIV-knockout cells while enhanced in Fut8- knockout cells. Furthermore, we assessed the expression levels of specific antibodies against ovalbumin and found they were downregulated in Fut8+/- mice, with potential recovery observed with L-fucose administration. These findings confirm that core fucosylation plays a vital role in regulating IgG levels in serum, which may provide insights into a novel mechanism in adaptive immune regulation. [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]

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]

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]

Uveal melanoma is the most frequent intraocular tumor, with a high mortality rate due to metastases, especially liver metastases. Glycosylation represents an enzymatic process of addition of molecules of saccharides to other saccharides, lipids or proteins. Aberrant glycosylation is considered a key feature in malignant transformation and cancer progression. In uveal melanoma, the most common glycosylation changes are considered sialylation, fucosylation, and N- and I-glycan branching. The goal of this study is to identify sialic acid as a tumoral marker and the correlation with survival rate in uveal malign melanoma. The determination of sialic acid is used in other cancers to monitor the evolution of the disease and the survival rate. Sialic acid specificity is relatively low, because high levels of sialic acid-rich glycoproteins are encountered in inflammatory diseases [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]

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]

Cancer secretome is a reservoir for aberrant glycosylation. How therapies alter this post- translational cancer hallmark and the consequences thereof remain elusive. Here, we show that an elevated secretome fucosylation is a pan-cancer signature of both response and resistance to multiple targeted therapies. Large-scale pharmacogenomics revealed that fucosylation genes display widespread association with resistance to these therapies. In cancer cell cultures, xenograft mouse models, and patients, targeted kinase inhibitors distinctively induced core fucosylation of secreted proteins less than 60 kDa. Label-free proteomics of N-glycoproteomes identified fucosylation of the antioxidant PON1 as a critical component of the therapy-induced secretome (TIS). N-glycosylation of TIS and target core fucosylation of PON1 are mediated by the fucose salvage-FUT8-SLC35C1 axis with PON3 directly modulating GDP-Fuc transfer on PON1 scaffolds. Core fucosylation in the Golgi impacts PON1 stability and folding prior to secretion, promoting a more degradation-resistant PON1. Global and PON1-specific secretome de-N-glycosylation both limited the expansion of resistant clones in a tumor regression model. We defined the resistance-associated transcription factors (TFs) and genes modulated by the N-glycosylated TIS via a focused and transcriptome-wide analyses. These genes characterize the oxidative stress, inflammatory niche, and unfolded protein response as important factors for this modulation. Our findings demonstrate that core fucosylation is a common modification indirectly induced by targeted therapies that paradoxically promotes resistance.

Abstract 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 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.

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]

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]

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 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]

Abstract Fucosyltransferases are enzymes that transfer L-fucose residues from a donor substrate to target molecules. These enzymes are encoded by genes known as FUTs (FUT1 to FUT-11), along with POFUT1 and 2. Changes in FUT expression have a significant role in cancer development and malignancy. This review delves into the biochemistry and biological functions of FUTs and their contributions to cancer. Broadly, FUTs play roles in cancer tumorigenesis, survival, and metastasis. Interactions between fucosylated glycans and various molecules associated with cancer, such as E-selectins and the epidermal growth factor receptor (EGFR), offer alternative pathways for cancer development. The review also highlights FUTs as potential biomarkers for cancer prognosis and diagnosis, along with their application as targets for therapy.

Human milk oligosaccharides (HMOs) are very important components in human milk, and more than 200 different HMOs have been identified so far. HMOs act as prebiotics by the metabolism of gut microbiota to produce short-chain fatty acids, which have a beneficial effect on infant health. This is one of the main differences between breast milk and formula milk powder. Studies have shown that different species of gut microbiota use different mechanisms to recognize and digest HMOs with different structures, maintaining the balance of the gut microbiota. HMOs also have a variety of functions such as relieving allergic symptoms and preventing necrotizing enterocolitis. In this paper, the type, structure and content of HMOs, interaction between HMOs and infant gut microbiota, and their immune regulatory functions are reviewed. HMOs can promote colonization and growth of gut microbiota in infants, and directly or indirectly regulate the infant immune function. This paper provides theoretical support for the application of HMOs in infant foods, and contributes new ideas to the future research and development direction.

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.

[Display omitted] [ABSTRACT FROM AUTHOR]

Abstract 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.

Long since, carbohydrates were thought to be used just as an energy source and structural material. However, in recent years, with the emergence of the field of glycobiology and advances in glycomics, much has been learned about the biological role of oligosaccharides, a carbohydrate polymer containing a small number of monosaccharides, in cell–cell interaction, signal transduction, immune response, pathogen adhesion processes, early embryogenesis, and apoptosis. The function of oligosaccharides in these processes is diversified by fucosylation, also known as modification of oligosaccharides. Fucosylation has allowed the identification of more than 100 different oligosaccharide structures that provide functional diversity. ABO blood group and Lewis antigens are among the best known fucosyl-linked oligosaccharides. In addition, the antigens in the ABO system are composed of various sugar molecules, including fucosylated oligosaccharides, and Lewis antigens are structurally similar to ABO antigens but differ in the linkage of sugars. Variation in blood group antigen expression affects the host’s susceptibility to many infections. However, altered expression of ABO and Lewis antigens is related with prognosis in carcinoma types. In addition, many pathogens recognize and bind to human tissues using a protein receptor with high affinity for the fucose molecule in glycoconjugates, such as lectin. Fucosylated oligosaccharides also play vital roles during fertilization and early embryogenesis. Learning and memory-related processes such as neurite growth, neurite migration, and synapse formation seen during the development of the brain, which is among the first organs to develop in embryogenesis, are regulated by fucosylated oligosaccharides. In conclusion, this review mentions the vital roles of fucosylated oligosaccharides in biology, drawing attention to their importance in the development of chemical tools to be used in function analysis and the investigation of various therapeutic targets.

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]

Histidine-rich glycoprotein (HRG) is an abundant plasma protein harboring at least three N-glycosylation sites. HRG integrates many biological processes, such as coagulation, antiangiogenic activity, and pathogen clearance. Importantly, HRG is known to exhibit five genetic variants with minor allele frequencies of more than 10%. Among them, Pro204Ser can induce a fourth N-glycosylation site (Asn202). Considerable efforts have been made to reveal the biological function of HRG, whereas data on HRG glycosylation are scarcer. To close this knowledge gap, we used C18-based LC-MS/MS to study the glycosylation characteristics of six HRG samples from different sources. We used endogenous HRG purified from human plasma and compared its glycosylation to that of the recombinant HRG produced in Chinese hamster ovary cells or human embryonic kidney 293 cells, targeting distinct genotypic isoforms. In endogenous plasma HRG, every N-glycosylation site was occupied predominantly with a sialylated diantennary complex-type glycan. In contrast, in the recombinant HRGs, all glycans showed different antennarities, sialylation, and core fucosylation, as well as the presence of oligomannose glycans, LacdiNAcs, and antennary fucosylation. Furthermore, we observed two previously unreported O-glycosylation sites in HRG on residues Thr273 and Thr274. These sites together showed more than 90% glycan occupancy in all HRG samples studied. To investigate the potential relevance of HRG glycosylation, we assessed the plasmin-induced cleavage of HRG under various conditions. These analyses revealed that the sialylation of the N- and O-glycans as well as the genotypedependent N-glycosylation significantly influenced the kinetics and specificity of plasmin-induced cleavage of HRG. [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]

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]

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.)

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]

Copyright of Brazilian Journal of Biology is the property of Instituto Internacional de Ecologia 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.)