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[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]

Proteinuria is a clinical manifestation of chronic kidney disease that aggravates renal interstitial fibrosis (RIF), in which injury of peritubular microvessels is an important event. However, the changes in peritubular microvessels induced by proteinuria and their molecular mechanisms remain unclear. Thus, we aimed to develop a co-culture microfluidic device that contains renal tubules and peritubular microvessels to create a proteinuria model. We found that protein overload in the renal tubule induced trans-differentiation and apoptosis of endothelial cells (ECs) and pericytes. Moreover, profiling of secreted proteins in this model revealed that a paracrine network between tubules and microvessels was activated in proteinuria-induced microvascular injury. Multiple cytokine receptors in this paracrine network were core-fucosylated. Inhibition of core fucosylation significantly reduced ligand-receptor binding ability and blocked downstream pathways, alleviating trans-differentiation and apoptosis of ECs and pericytes. Furthermore, the protective effect of genetic FUT8 deficiency on proteinuria overload-induced RIF and pericyte-myofibroblast trans-differentiation was validated in FUT8 knockout heterozygous mice. In conclusion, we constructed and used a multiple-unit integrated microfluidic device to uncover the mechanism of proteinuria-induced RIF. Furthermore, FUT8 may serve as a hub-like therapeutic target to alleviate peritubular microvascular injury in RIF. In this study, we constructed a multiple-unit integrated renal tubule-vascular chip. We reproduced human proteinuria on the chip and found that multiple receptors were modified by FUT8-catalyzed core fucosylation (CF) involved in the cross-talk between renal tubules and peritubular microvessels in proteinuria-induced RIF, and inhibiting the FUT8 of receptors could block the tubule-microvessel paracrine network and reverse the damage of peritubular microvessels and renal interstitial fibrosis. This tubule-vascular chip may provide a prospective platform to facilitate future investigations into the mechanisms of kidney diseases, and target-FUT8 inhibition may be an innovative and potential therapeutic strategy for RIF induced by proteinuria. [Display omitted] [ABSTRACT FROM AUTHOR]

• Fut8 is dramatically up-regulated from DN to DP transition during thymic development. • Core fucosylation is required for the signal transduction via TCR. • Loss of Fut8 results in the reduced CD4+ SP, CD8+ SP and DP cell population. • Loss of Fut8 reduces positive selection during the thymic development. Precise glycosylation plays a crucial and distinctive role in thymic T cell development. The core fucosylation is dramatically up-regulated at the transition from CD4−CD8− (DN) to CD4+CD8+ (DP) in the thymic development. Ablation of core fucosylation in T cells did reduce the size of the thymus due to a significant loss of CD4+ SP, CD8+ SP and DP thymocytes in core fucosyltransferase (Fut8) knockout (Fut8−/−) mice. T cell receptors (TCRs) are heavily core fucosylated glycoproteins. Loss of core fucosylation of TCR contributed to the reduced phosphorylation of ZAP70 (pZAP70) in Fut8−/− DP cells was observed. Compare to the Fut8+/+OT-II DP thymocytes, pZAP70 was significantly reduced in Fut8−/− OT-II DP thymocytes with OVA 323–339 stimulation. Also, the pZAP70 of Fut8+/+OT-I DP thymocytes with OVA 257–264 stimulation was remarkably attenuated by treatment of the fucosidase. Upon anti-CD3/CD28 Abs stimulation, the increased apoptosis was found in Fut8−/− thymocytes compared with Fut8+/+ thymocytes. Moreover, the TCRhiCD69hi (post-positive selection thymocytes) was markedly depleted in the Fut8−/− thymus without any stimulation. The expression of CD5 was significantly down-regulated on the DP cells in the Fut8−/− thymus. Our results therefore demonstrate that ablation of core fucosylation results in the abnormal T cell development due to the attenuated signaling via TCR. [ABSTRACT FROM AUTHOR]

Abstract Background α1,6-Fucosyltransferase-deficient (Fut8−/−) mice displayed increased locomotion and schizophrenia-like behaviors. Since neuroinflammation is a common pathological change in most brain diseases, this study was focused on investigating the effects of Fut8 in microglia and astrocytes. Methods Brain tissues were analyzed using immunohistochemical staining. Core fucosylation and protein expression were analyzed using lectin blot and western blot, respectively. Fut8-knockout (KO) cells were established by the CRISPR/Cas9 system. Results The number of Iba-1 positive cells and GFAP positive cells were significantly increased in both untreated and lipopolysaccharide stimulated inflammatory conditional Fut8−/− mice by comparison with both wild-type (Fut8+/+) and hetero (Fut8+/−) mice. Stimulation with pro-inflammatory factors, such as IFN-γ and IL-6, induced expression levels of fucosylation in primary microglia and astrocytes, as well as in glial cell lines. Cell motility and iNOS expression were easily induced by IFN-γ in Fut8-KO BV-2 cells compared with wild-type (WT) cells. In a similar manner, both Fut8-KO C6 cells and primary astrocytes treated with 2-fluoro-L-fucose, a specific inhibitor for fucosylation, showed a higher response to IL-6-stimulated phospho-STAT3 signaling, compared with WT cells. Conclusions Core fucosylation negatively regulates the states of neuroinflammation by modulating the sensitivity of microglia and astrocytes to inflammatory mediators. The disorders of Fut8−/− mice are caused not only by neurons but also by glial cell dysfunction. General significance Core fucose is a novel regulator for neuroinflammation in the central nervous system. Highlights • Core fucosylation is a novel regulator for neuroinflammation and glial cell activation. • Decreased core fucosylation could increase the sensitivity of glial cells to inflammatory mediators. • Core fucosylation differently regulates the pro-inflammatory and anti-inflammatory signaling. [ABSTRACT FROM AUTHOR]

Abstract Aberrant core fucosylation of proteins has been linked to liver diseases. In this study, we carried out multiple reaction monitoring (MRM) quantification of core fucosylated N-glycopeptides of serum proteins partially deglycosylated by a combination of endoglycosidases (endoF1, endoF2, and endoF3). To minimize variability associated with the preparatory steps, the analysis was performed without enrichment of glycopeptides or fractionation of serum besides the nanoRP chromatography. Specifically, we quantified core fucosylation of 22 N-glycopeptides derived from 17 proteins together with protein abundance of these glycoproteins in a cohort of 45 participants (15 disease-free control, 15 fibrosis and 15 cirrhosis patients) using a multiplex nanoUPLC-MS-MRM workflow. We find increased core fucosylation of 5 glycopeptides at the stage of liver fibrosis (i.e., N630 of serotransferrin, N107 of alpha-1-antitrypsin, N253 of plasma protease C1 inhibitor, N397 of ceruloplasmin, and N86 of vitronectin), increase of additional 6 glycopeptides at the stage of cirrhosis (i.e., N138 and N762 of ceruloplasmin, N354 of clusterin, N187 of hemopexin, N71 of immunoglobulin J chain, and N127 of lumican), while the degree of core fucosylation of 10 glycopeptides did not change. Interestingly, although we observe an increase in the core fucosylation at N86 of vitronectin in liver fibrosis, core fucosylation decreases on the N169 glycopeptide of the same protein. Our results demonstrate that the changes in core fucosylation are protein and site specific during the progression of fibrotic liver disease and independent of the changes in the quantity of N-glycoproteins. It is expected that the fully optimized multiplex LC-MS-MRM assay of core fucosylated glycopeptides will be useful for the serologic assessment of the fibrosis of liver. Biological significance We have quantified the difference in core fucosylation among three comparison groups (healthy control, fibrosis and cirrhosis patients) using a sensitive and selective LC-MS-MRM method. Despite an overall increase in core fucosylation of many of the glycoproteins that we examined, core fucosylation changed in a protein- and site-specific manner. Moreover, increased and decreased fucosylation was observed on different N-glycopeptides of the same protein. Altered core fucosylation of N-glycopeptides might be used as an alternative serologic assay for the evaluation of fibrotic liver disease. Graphical abstract Unlabelled Image Highlights • A LC-MS-MRM-method was established for quantifying core focusylation of serum proteins • Core fucosylation was compared between control, liver fibrosis and cirrhosis participants • Core fucosylation occurred in a site-specific and protein-specific manner [ABSTRACT FROM AUTHOR]

Summary Programmed cell death 1 (PD-1) is highly expressed on exhausted T cells and inhibits T cell activation. Antibodies that block the interaction between PD-1 and its ligand prevent this inhibitory signal and reverse T cell dysfunction, providing beneficial anti-tumor responses in a substantial number of patients. Mechanisms for the induction and maintenance of high PD-1 expression on exhausted T cells have not been fully understood. Utilizing a genome-wide loss-of-function screening method based on the CRISPR-Cas9 system, we identified genes involved in the core fucosylation pathway as positive regulators of cell-surface PD-1 expression. Inhibition of Fut8, a core fucosyltransferase, by genetic ablation or pharmacologic inhibition reduced cell-surface expression of PD-1 and enhanced T cell activation, leading to more efficient tumor eradication. Taken together, our findings suggest that blocking core fucosylation of PD-1 can be a promising strategy for improving anti-tumor immune responses. [ABSTRACT FROM AUTHOR]

The levels of core fucosylation and α2,3-linked sialic acid in serum Prostate Specific Antigen (PSA), using the lectins Pholiota squarrosa lectin (PhoSL) and Sambucus nigra agglutinin (SNA), can discriminate between Benign Prostatic Hyperplasia (BPH) and indolent prostate cancer (PCa) from aggressive PCa. In the present work we evaluated whether these glycosylation determinants could also be altered in urinary PSA obtained after digital rectal examination (DRE) and could also be useful for diagnosis determinations. For this purpose, α2,6-sialic acid and α1,6-fucose levels of urinary PSA from 53 patients, 18 biopsy-negative and 35 PCa patients of different aggressiveness degree, were analyzed by sandwich ELLA (Enzyme Linked Lectin Assay) using PhoSL and SNA. Changes in the levels of specific glycosylation determinants, that in serum PSA samples were indicative of PCa aggressiveness, were not found in PSA from DRE urine samples. Although urine is a simpler matrix for analyzing PSA glycosylation compared to serum, an immunopurification step was necessary to specifically detect the glycans on the PSA molecule. Those specific glycosylation determinants on urinary PSA were however not useful to improve PCa diagnosis. This could be probably due to the low proportion of PSA from the tumor in urine samples, which precludes the identification of aberrantly glycosylated PSA. [ABSTRACT FROM AUTHOR]

Pulmonary fibrosis is a common outcome of a variety of pulmonary interstitial diseases, and myofibroblasts are the main culprit for this process. Recent studies have found that pericytes are one of the major sources of myofibroblasts; the transformation of which involves a complex process of activation of TGF-β/Smad2/3 and PDGFβ/Erk signaling pathways. We have reported that the transforming growth factor-β receptor and platelet-derived growth factor-β receptor (TGF-βR I and PDGFβR, respectively) are modified by glycosylation. Thus, we hope to regulate the above-mentioned signal pathways through core fucosylation (CF) catalyzed by α-1,6-fucosyltransferase (FUT8). Previous work has confirmed that TGF-β1 can induce the transformation of pericytes into myofibroblasts, while FUT8siRNA can inhibit such transformation. In the present study, we used an adenovirus packaging FUT8 shRNA to infect a bleomycin-induced pulmonary fibrosis mouse model and determined the effect of CF on pulmonary fibrosis by analyzing the mechanism of CF-mediated pericyte transformation. Our findings may shed new light on the mechanism of pulmonary interstitial fibrosis and provide a novel therapeutic target for clinical applications. [ABSTRACT FROM AUTHOR]

Abstract: Comprehensive analyses of proteins from cells and tissues are the most effective means of elucidating the expression patterns of individual disease-related proteins. On the other hand, the simultaneous separation and characterization of proteins by 1-DE or 2-DE followed by MS analysis are one of the fundamental approaches to proteomic analysis. However, these analyses do not permit the complete structural identification of glycans in glycoproteins or their structural characterization. Over half of all known proteins are glycosylated and glycan analyses of glycoproteins are requisite for fundamental proteomics studies. The analysis of glycan structural alterations in glycoproteins is becoming increasingly important in terms of biomarkers, quality control of glycoprotein drugs, and the development of new drugs. However, usual approach such as proteoglycomics, glycoproteomics and glycomics which characterizes and/or identifies sugar chains, provides some structural information, but it does not provide any information of functionality of sugar chains. Therefore, in order to elucidate the function of glycans, functional glycomics which identifies the target glycoproteins and characterizes functional roles of sugar chains represents a promising approach. In this review, we show examples of functional glycomics technique using α 1,6 fucosyltransferase gene (Fut8) in order to identify the target glycoprotein(s). This approach is based on glycan profiling by CE/MS and LC/MS followed by proteomic approaches, including 2-DE/1-DE and lectin blot techniques and identification of functional changes of sugar chains. [Copyright &y& Elsevier]

[Display omitted] Core fucosylation is the attachment of an α-1,6-fucose moiety to the innermost N -acetyl glucosamine (GlcNAc) in N -glycans in mammalian systems. It plays a pivotal role in modulating the structural and biological functions of glycoproteins including therapeutic antibodies. Yet, few α- l -fucosidases appear to be capable of removing core fucose from intact glycoproteins. This paper describes a comparative study of the substrate specificity and relative activity of the human α- l -fucosidase (FucA1) and two bacterial α- l -fucosidases, the AlfC from Lactobacillus casei and the BfFuc from Bacteroides fragilis. This study was enabled by the synthesis of an array of structurally well-defined core-fucosylated substrates, including core-fucosylated N -glycopeptides and a few antibody glycoforms. It was found that AlfC and BfFuc could not remove core fucose from intact full-length N -glycopeptides or N -glycoproteins but could hydrolyze only the truncated Fucα1,6GlcNAc-peptide substrates. In contrast, the human α- l -fucosidase (FucA1) showed low activity on truncated Fucα1,6GlcNAc substrates but was able to remove core fucose from intact and full-length core-fucosylated N -glycopeptides and N -glycoproteins. In addition, it was found that FucA1 was the only α- l -fucosidase that showed low but apparent activity to remove core fucose from intact IgG antibodies. The ability of FucA1 to defucosylate intact monoclonal antibodies reveals an opportunity to evolve the human α- l -fucosidase for direct enzymatic defucosylation of therapeutic antibodies to improve their antibody-dependent cellular cytotoxicity. [ABSTRACT FROM AUTHOR]

Pancreatic carcinoma is one of the deadliest malignant diseases, in which the increased expression of α1,6-fucosyltransferase (FUT8), a sole enzyme responsible for catalyzing core fucosylation, has been reported. However, its pathological roles and regulatory mechanisms remain largely unknown. Here, we use two pancreatic adenocarcinoma cell lines, MIA PaCa-2 and PANC-1 cells, as cell models, to explore the relationship of FUT8 with the malignant transformation of PDAC. FUT8 knockout (FUT8-KO) cells were established by the CRISPR/Cas9 system. Cell migration was analyzed by transwell and wound-healing assays. Cell proliferation was examined by MTT and colony-formation assays. Cancer stemness markers and spheroid formations were used to analyzed cancer stemness features. Deficiency of FUT8 inhibited cell migration and proliferation in both MIA PaCa-2 and PANC-1 cells compared with wild-type cells. Moreover, the expression levels of cancer stemness markers such as EpCAM, CXCR4, c-Met, and CD133 were decreased in the FUT8-KO cells compared with wild-type cells. Also, the spheroid formations in the KO cells were loose and unstable, which could be reversed by restoration with FUT8 gene in the KO cells. Additionally, FUT8-KO increased the chemosensitivity to gemcitabine, which is the first-line therapy for advanced pancreatic cancer. FUT8-KO reduced the cell proliferation and migration. Our results are the first to suggest that the expression of FUT8 is involved in regulating the stemness features of pancreatic cancer cells. FUT8 could provide novel insights for the treatment of pancreatic carcinoma. [ABSTRACT FROM AUTHOR]

Previous structural analyses showed that human α1,6-fucosyltransferase, FUT8 contains a catalytic domain along with two additional domains, N-terminal α-helical domain and C-terminal Src homology 3 domain, but these domains are unique to FUT8 among glycosyltransferases. The role that these domains play in formation of the active form of FUT8 has not been investigated. This study reports on attempts to determine the involvement of these domains in the functions of FUT8. Based on molecular modeling, the domain mutants were constructed by truncation and site-directed mutagenesis, and were heterologously expressed in Sf21 or COS-1 cells. The mutants were analyzed by SDS-PAGE and assayed for enzymatic activity. In vivo cross-linking experiments by introducing disulfide bonds were also carried out to examine the orientation of the domains in the molecular assembly. Mutagenesis and molecular modeling findings suggest that human FUT8 potentially forms homodimer in vivo via intermolecular hydrophobic interactions involving α-helical domains. Truncation or site-directed mutagenesis findings indicated that α-helical and SH3 domains are all required for enzymatic activity. In addition, in vivo cross-linking experiments clearly indicated that the SH3 domain located in close proximity to the α-helical domain in an intermolecular manner. α-Helical and SH3 domains are required for a fully active enzyme, and are also involved in homophilic dimerization, which probably results in the formation of the active form of human FUT8. α-Helical and SH3 domains, which are not commonly found in glycosyltransferases, play roles in the formation of the functional quaternary structure of human FUT8. • Human α1,6-fucosyltransferase, FUT8 was proposed to be a homodimer. • α-Helical and SH3 domains are required for the enzymatic activity of FUT8. • The hydrophobic interaction of α-helical domain is involved in the dimerization. • The spatial orientation of SH3 domain is involved in the dimerization of FUT8. [ABSTRACT FROM AUTHOR]

Summary Association of aberrant glycosylation with melanoma progression is based mainly on analyses of cell lines. Here we present a systems-based study of glycomic changes and corresponding enzymes associated with melanoma metastasis in patient samples. Upregulation of core fucosylation (FUT8) and downregulation of α-1,2 fucosylation (FUT1, FUT2) were identified as features of metastatic melanoma. Using both in vitro and in vivo studies, we demonstrate FUT8 is a driver of melanoma metastasis which, when silenced, suppresses invasion and tumor dissemination. Glycoprotein targets of FUT8 were enriched in cell migration proteins including the adhesion molecule L1CAM. Core fucosylation impacted L1CAM cleavage and the ability of L1CAM to support melanoma invasion. FUT8 and its targets represent therapeutic targets in melanoma metastasis. [ABSTRACT FROM AUTHOR]