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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]

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]

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]

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]

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]

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]

The combination of helium charge transfer dissociation mass spectrometry (He–CTD–MS) with ultrahigh performance liquid chromatography (UHPLC) is presented for the analysis of a complex mixture of acidic and neutral human milk oligosaccharides (HMOs). The research focuses on the identification of the monosaccharide sequence, the branching patterns, the sialylation/fucosylation arrangements, and the differentiation of isomeric oligosaccharides in the mixture. Initial studies first optimized the conditions for the UHPLC separation and the He–CTD–MS conditions. Results demonstrate that He–CTD is compatible with UHPLC timescales and provides unambiguous glycosidic and cross-ring cleavages from both the reducing and the nonreducing ends, which is not typically possible using collision-induced dissociation. He–CTD produces informative fragments, including 0,3An and 0,4An ions, which have been observed with electron transfer dissociation, electron detachment dissociation, and ultraviolet photodissociation (UVPD) and are crucial for differentiating the α-2,3- versus α-2,6-linked sialic acid (Neu5Ac) residues present among sialyllacto- N -tetraose HMOs. In addition to the linkage positions, He–CTD is able to differentiate structural isomers for both sialyllacto- N -tetraoses and lacto- N -fucopentaoses structures by providing unique, unambiguous cross-ring cleavages of types 0,2An, 0,2Xn, and 1,5An while preserving most of the labile Neu5Ac and fucose groups. [ABSTRACT FROM AUTHOR]

Maturity-onset diabetes of the young due to hepatocyte nuclear factor-1 alpha variants (HNF1A-MODY) causes monogenic diabetes. Individuals carrying damaging variants in HNF1A show decreased levels of α1-3,4 fucosylation, as demonstrated on antennary fucosylation of blood plasma N -glycans. The excellent diagnostic performance of this glycan biomarker in blood plasma N -glycans of individuals with HNF1A-MODY has been demonstrated using liquid chromatography methods. Here, we have developed a high-throughput exoglycosidase plate-based assay to measure α1-3,4 fucosylation levels in blood plasma samples. The assay has been optimized and its validity tested using 1000 clinical samples from a cohort of individuals with young-adult onset diabetes including cases with HNF1A-MODY. The α1-3,4 fucosylation levels in blood plasma showed a good differentiating power in identifying cases with damaging HNF1A variants, as demonstrated by receiver operating characteristic curve analysis with the AUC values of 0.87 and 0.95. This study supports future development of a simple diagnostic test to measure this glycan biomarker for application in a clinical setting. [ABSTRACT FROM AUTHOR]

The α(1,6)fucose residue attached to the N -glycoprotein core is suspected to play an essential role in the progression of several types of cancer. Lectins remain the first choice for probing glycan modifications, although they may lack specificity. Thus, efforts have been made to identify new lectins with a narrower core fucose (CF) detection profile. Here, we present a comparison of the classical Aleuria aurantia lectin (AAL), Lens culinaris agglutinin (LCA) and Aspergillus oryzae lectin (AOL) with the newer Pholiota squarrosa lectin (PhoSL), which has been described as being specific for core fucosylated N -glycans. To this end, we studied the binding profiles of the four lectins using mammalian glycan arrays from the Consortium of Functional Glycomics. To validate their glycan specificity, we probed AOL, LCA and PhoSL in western-blot assays using protein extracts from eight common colorectal cancer (CRC) lines and colorectal biopsies from a small cohort of patients with CRC. The results showed that (i) LCA and PhoSL were the most specific lectins for detecting the presence of CF in a concentration-dependent manner; (ii) PhoSL exhibited the highest N -glycan sequence restriction, with preferential binding to core fucosylated paucimannosidic-type N -glycans, (iii) the recognition ability of PhoSL was highly influenced by the presence of terminal N -acetyl-lactosamine; (iv) LCA bound to paucimannosidic, bi-antennary and tri-antennary core fucosylated N -glycans and (v) AOL and AAL exhibited broader specificity towards fucosylation. Together, our results support the choice of LCA as the most appropriate lectin for CF detection, as validated in protein extracts from CRC cell lines and tissue specimens from patients with CRC. [ABSTRACT FROM AUTHOR]

Pseudomyxoma peritonei (PMP) is a highly mucinous adenocarcinoma growing in the peritoneal cavity and most commonly originating from the appendix. Glycans play an important role in carcinogenesis, and glycosylation is altered in malignant diseases, including PMP. We have previously demonstrated that fucosylation of N-glycans is increased in PMP, but we did not observe modulation of overall sialylation. As sialic acids can be attached to the rest of the glycan via α2,3- or α2,6-linkage, we have now analyzed the linkage patterns of sialic acids in tissue specimens of normal appendices, low-grade appendiceal mucinous neoplasms (LAMN), low-grade (LG) PMP and high-grade (HG) PMP. For the linkage analysis, the enzymatically released acidic N-glycans were first treated with ethyl esterification or α2,3-sialidase digestion followed by MALDI-TOF mass spectrometry. Significant increase in the relative abundance of α2,6-sialylated and decrease in α2,3-sialylated N-glycans was observed in PMP tumors as compared to the normal appendices (P  < 0.025). More specifically, increased α2,6-sialylation (P  < 0.05) and decreased α2,3-sialylation (P  < 0.01) were detected in afucosylated and monofucosylated N-glycans of PMPs, whereas the less abundant multifucosylated glycans, containing terminal fucose, demonstrated increased α2,3-sialylation (P  < 0.01). Importantly, the increase in α2,6-sialylation was also detected between PMP and the appendiceal precursor lesion LAMN (P  < 0.01). The identified glycosylation alterations produce ligands for sialic acid-binding immunoglobulin-like lectins (Siglecs) and sialofucosylated glycans binding selectins, which play a role in the peritoneal dissemination and progression of the disease. [ABSTRACT FROM AUTHOR]

Like sialylation, fucose usually locates at the nonreducing ends of various glycans on glycoproteins and constitutes important glycan epitopes. Detecting the substrate glycans of fucosyltransferases is important for understanding how these glycan epitopes are regulated in response to different growth conditions and external stimuli. Here we report the detection of these glycans on glycoproteins as well as in their free forms via enzymatic incorporation of fluorophore-conjugated fucose using FUT2, FUT6, FUT7, FUT8 and FUT9. Specifically, we describe the detection of the substrate glycans of these enzymes on fetal bovine fetuin, recombinant H1N1 viral neuraminidase and therapeutic antibodies. The detected glycans include complex and high-mannose N-glycans. By establishing a series of precursors for the synthesis of Lewis X and sialyl Lewis X structures, we not only provide convenient electrophoresis methods for studying glycosylation but also demonstrate the substrate specificities and some kinetic features of these enzymes. Our results support the notion that fucosyltransferases are key targets for regulating the synthesis of Lewis X and sialyl Lewis X structures. [ABSTRACT FROM AUTHOR]

Protein glycosylation impacts the development and function of innate immune cells. The glycophenotypes and the glycan remodelling associated with the maturation of macrophages from monocytic precursor populations remain incompletely described. Herein, label-free porous graphitised carbon–liquid chromatography–tandem mass spectrometry (PGC-LC-MS/MS) was employed to profile with high resolution the N - and O -glycome associated with human monocyte-to-macrophage transition. Primary blood-derived CD14+ monocytes were differentiated ex vivo in the absence of strong anti- and proinflammatory stimuli using a conventional 7-day granulocyte-macrophage colony-stimulating factor differentiation protocol with longitudinal sampling. Morphology and protein expression monitored by light microscopy and proteomics validated the maturation process. Glycomics demonstrated that monocytes and macrophages display similar N -glycome profiles, comprising predominantly paucimannosidic (Man1-3GlcNAc2Fuc0–1, 22.1–30.8%), oligomannosidic (Man5-9GlcNAc2, 29.8–35.7%) and α2,3/6-sialylated complex-type N -glycans with variable core fucosylation (27.6–39.1%). Glycopeptide analysis validated conjugation of these glycans to human proteins, while quantitative proteomics monitored the glycoenzyme expression levels during macrophage differentiation. Significant interperson glycome variations were observed suggesting a considerable physiology-dependent or heritable heterogeneity of CD14+ monocytes. Only few N -glycome changes correlated with the monocyte-to-macrophage transition across donors including decreased core fucosylation and reduced expression of mannose-terminating (paucimannosidic-/oligomannosidic-type) N -glycans in macrophages, while lectin flow cytometry indicated that more dramatic cell surface glycan remodelling occurs during maturation. The less heterogeneous core 1-rich O -glycome showed a minor decrease in core 2-type O -glycosylation but otherwise remained unchanged with macrophage maturation. This high-resolution glycome map underpinning normal monocyte-to-macrophage transition, the most detailed to date, aids our understanding of the molecular makeup pertaining to two vital innate immune cell types and forms an important reference for future glycoimmunological studies. [ABSTRACT FROM AUTHOR]

IgG glycosylation is currently at the forefront of both immunology and glycobiology, likely due in part to the widespread and growing use of antibodies as drugs. For over four decades, it has been recognized that the conserved N-linked glycan on asparagine 297 found within the second Ig domain of the heavy chain (CH2) that helps to comprise Fc region of IgG plays a special role in IgG structure and function. Changes in galactosylation, fucosylation and sialylation are now well-established factors, which drive differential IgG function, ranging from inhibitory/anti-inflammatory to activating complement and promoting antibody-dependent cellular cytotoxicity. Thus, if we are to truly understand how to design and deploy antibody-based drugs with maximal efficacy and evaluate proper vaccine responses from a protective and functional perspective, a deep understanding of IgG glycosylation is essential. This article is intended to provide a comprehensive review of the IgG glycosylation field and the impact glycans have on IgG function, beginning with the earliest findings over 40 years ago, in order to provide a robust foundation for moving forward. [ABSTRACT FROM AUTHOR]

Certain viral infections are known to modify the glycosylation profile of infected cells through the overexpression of specific host cell fucosyltransferases (FUTs). Infection with CMV (cytomegalovirus), HCV (hepatitis C virus), HSV-1 (herpes simplex virus type-1) and VZV (varicella-zoster virus) increase the expression of fucosylated epitopes, including antigens sLex (Siaα2–3 Galβ1–4(Fucα1–3)GlcNAcβ1-R) and Ley (Fucα1–2 Galβ1–4(Fucα1–3)GlcNAcβ1-R). The reorganization of the glycocalyx induced by viral infection may favor the spread of viral progeny, and alter diverse biological functions mediated by glycans, including recognition by the adaptive immune system. In this work, we aimed to establish whether infection with human adenovirus type 5 (HAd5), a well-known viral vector and infectious agent, causes changes in the glycosylation profile of A549 cells, used as a model of lung epithelium, a natural target of HAd5. We demonstrate for the first time that HAd5 infection causes a significant increase in the cell surface de novo fucosylation, as assessed by metabolic labeling, and that such modification is dependent on the expression of viral genes. The main type of increased fucosylation was determined to be in α1–2 linkage, as assessed by UEA-I lectin binding and supported by the overexpression of FUT1 and FUT2. Also, HAd5-infected cells showed a heterogeneous change in the expression profile of the bi-fucosylated Ley antigen, an antigen associated with enhanced cell proliferation and inhibition of apoptosis. [ABSTRACT FROM AUTHOR]

One hundred human proteins have one or more EGF-like domains (EGF-LD) bearing the O -fucosylation consensus motif C2X4(S / T)C3 but to date, only a few of them have been shown to be O -fucosylated. The protein O -fucosyltransferase (POFUT1) specifically recognizes correctly folded EGF-LD of the human EGF (hEGF) type and transfers fucose on serine or threonine residue within the O -fucosylation motif. Here, we propose a strategy for a rapid screening for ability of any EGF-LD to be O -fucosylated, using copper-catalyzed azide-alkyne cycloaddition (CuAAC). By an oligonucleotide hybridization approach, double-stranded fragments encoding any EGF-LD can be first rapidly cloned into the prokaryotic vector pET-25b to promote its targeting to periplasm and formation of the three conserved disulfide bonds. After protein production and purification, an in vitro POFUT1-mediated O -fucosylation can be performed with azido GDP-fucose. Successful transfer of O -fucose is finally revealed by blotting after CuAAC. In this study, we specially focused on mouse NOTCH1 EGF12 and EGF26, which are both known to be O -fucosylated although having different binding affinities towards POFUT1. Indeed, we clearly showed here that addition of O -fucose by POFUT1 was much more efficient for EGF26 than for EGF12. This experimental approach is rapid and sufficiently sensitive to reveal propensity of any EGF-LD to be O -fucosylated; it is thus useful prior to perform structure–function studies on target proteins containing one or several EGF-LD. [ABSTRACT FROM AUTHOR]

The hepatitis B virus (HBV)-induced chronic liver diseases are serious health threats worldwide. There is evidence to display the alterations of salivary N-linked glycans related to the development of HBV-infected liver diseases. Here, we further investigated the alterations of fucosylated N / O -glycans recognized by LTL in saliva from 120 subjects (30 healthy volunteers (HV), 30 patients with hepatitis B (HB), 30 patients with hepatic cirrhosis (HC), and 30 patients with hepatocellular carcinoma (HCC)) using salivary microarrys and MALDI-TOF/TOF-MS. The results showed that the expression level of fucosylated glycans recognized by LTL was significantly increased in HCC compared with other subjects (P < 0.0001). Besides, the fucosylated glycoproteins were isolated from pooled saliva of HV, HB, HC, and HCC by LTL-magnetic particle conjugates. Then, N / O - glycans were released from the isolated glycoproteins with PNGase F and NaClO, and were identified by MALDI-TOF-MS, respectively. Totally, there were 21/20, 25/18, 29/19, and 28/24 N / O -glycan peaks that were identified and annotated with proposed structures in saliva of HV, HB, HC, and HCC. Among the total, there were 8 N-glycan peaks (e.g. m / z 1905.634, 2158.777 and 2905.036) and 15 O -glycan peaks (e.g. 1177.407, 1308.444 and 1322.444) that only presented in patients with HBV-induced liver diseases. One N-glycan peak (m / z 2205.766) was unique in HC, and 9 O -glycan peaks (e.g. m / z 1157.420, 1163.417 and 1193.402) were unique in HCC. This study could facilitate the discovery of biomarkers for HC and HCC based on precise alterations of fucosylated N / O -glycans in saliva. [ABSTRACT FROM AUTHOR]

Fc glycosylation of human immunoglobulins G (IgGs) is essential for their structural integrity and activity. Interestingly, the specific nature of the Fc glycoforms is known to modulate the IgG effector function and inflammatory properties. Indeed, while core-fucosylation of IgG Fc-glycans greatly affects the antibody-dependent cell-mediated cytotoxicity function, with obvious repercussions in the design of therapeutic antibodies, sialylation can reverse the antibody inflammatory response, and galactosylation levels have been linked to aging, to the onset of inflammation, and to the predisposition to rheumatoid arthritis. Within the framework of a structure-to-function relationship, we have studied the role of the N -glycan sequence on its intrinsic conformational propensity. Here we report the results of a systematic study, based on extensive molecular dynamics simulations in excess of 62 μs of cumulative simulation time, on the effect of sequence on the structure and dynamics of increasingly larger, complex biantennary N-glycoforms isolated from the protein, i.e. from chitobiose to the larger N -glycan species commonly found in the Fc region of human IgGs. Our results show that while core fucosylation and sialylation do not affect the intrinsic dynamics of the unlinked N -glycans, galactosylation of the α(1–6) arm shifts dramatically its conformational equilibrium from an outstretched to a folded conformation. These findings are in agreement with and can help rationalize recent experimental evidence showing a differential recognition of positional isomers in glycan array data and also the preference of sialyltransferase for the more accessible, outstretched α(1–3) arm in both isolated, and Fc-bound N -glycans. [ABSTRACT FROM AUTHOR]

Abstract: Global antibody glycosylation is dynamic and plays critical roles in shaping different immunological outcomes and direct antibody functionality during HIV infection. However, the relevance of global antibody or plasma glycosylation patterns to HIV persistence after antiretroviral therapy (ART) has not been characterized. First, we compared glycomes of total plasma and isolated immunoglobulin G (IgG) from HIV+ ART‐suppressed, HIV+ viremic, and HIV‐negative individuals. Second, in ART‐suppressed individuals, we examined the associations between glycomes and (1) levels of cell‐associated HIV DNA and RNA in PBMCs and isolated CD4+ T cells, (2) CD4 count and CD4%, and (3) expression of CD4+ T‐cell activation markers. HIV infection is associated with persistent alterations in the IgG glycome including decreased levels of disialylated glycans, which is associated with a lower anti‐inflammatory activity, and increased levels of fucosylated glycans, which is associated with lower antibody‐dependent cell‐mediated cytotoxicity (ADCC). We also show that levels of certain mono‐ and digalactosylated nonfucosylated glycomic traits (A2G1, A2G2, and A2BG2), which have been reported to be associated with higher ADCC and higher anti‐inflammatory activities, exhibit significant negative correlations with levels of cell‐associated total HIV DNA and HIV RNA in ART‐suppressed individuals. Finally, levels of certain circulating anti‐inflammatory glycans are associated with higher levels of CD4 T cells and lower levels of T‐cell activation. Our findings represent the first proof‐of‐concept evidence that glycomic alterations, known to be associated with differential states of inflammation and ADCC activities, are also associated with levels of HIV persistence in the setting of ART suppression. [ABSTRACT FROM AUTHOR]

Fucosylated chondroitin sulfates (FCSs) and sulfated fucans (SFs) are conspicuous components of the body wall of sea cucumbers (Holothuroidea). FCSs are composed of a central core of chondroitin sulfate (CS) decorated with branches of mono- or both mono- and disaccharides of α-fucose (FCS types I and II, respectively). FCSs type II have heterogeneous and irregularly distributed α-fucose branches; however, the novel FCS type II from Holothuria lentiginosa described herein via solution nuclear magnetic resonance has strikingly homogeneous α-fucose branches neatly distributed along its CS core. This FCS is built up of three distinct sequential units composed of the typical CS disaccharides of FCSs, rich in β-galactosamine-4,6diS, decorated with branches of α-Fuc p -2,4diS, α-Fuc p -3,4diS or α-Fuc p [1→3]α-Fuc p -4S[1→ linked to the position 3- of the β-glucuronic acid. Conformational analyses of these repetitive units revealed a fairly rigid structure despite of the high sulfate content of their α-fucose branches. We also determined the structure of the SF from H. lentiginosa as a repetitive tetrasaccharide sequence composed of →3]α-Fuc p -2,4diS[1→3]α-Fuc p [1→3]α-Fuc p -2S[1→3]α-Fuc p -2S[1→. Furthermore, we determined that the nonsulfated α-fucose units present in FCS type II did not interfere with their anticoagulant potencies and affinities to calcium. FCS is an autapomorphic molecular character of the class Holothuroidea and the composition of their α-fucose branches differs in a species-specific manner. Branches containing α-Fuc p -2,4diS are the most common within the extant holothurians, being found in 90% of the FCSs characterized thus far. [ABSTRACT FROM AUTHOR]

Microbial catalysis has recently emerged as one of the most promising approaches in oligosaccharide synthesis. However, despite significant progress, microbial synthesis still requires much improvement in efficiency and in reduction of process complexity. Additionally, given the stunning diversity and many varied applications of glycans, broadening the range of glycans accessible via microbial synthesis is of paramount importance. Major challenges in microbial synthesis include catabolite repression and high cellular energy requirement. Here we demonstrated a new approach to overcome these challenges by directly tapping into the cellular "power house," the TCA cycle, to provide the cellular energy for synthesis. This approach not only circumvents catabolite repression but also eliminates acidic glycolysis by-products. As such, the whole-cell biocatalysis can be carried out without sophisticated fed-batch feeding and pH control in the synthesis stage. The system could achieve several grams per liter (3-4 g/L) within a 24-h period in shaker flask cultivation for two targets, fucosyllactose and fucosyllactulose, demonstrating efficiency of the biocatalyst developed and its applicability to both natural and non-natural targets. To the best of our knowledge, this is the first use of TCA cycle intermediates as the energy source for oligosaccharide synthesis and the first description of successful synthesis of fucosyllactulose with titers in several grams per liter. [ABSTRACT FROM AUTHOR]

Recently, the absence of a core-fucose residue in the N-glycan has been implicated to be important for enhancing antibody-dependent cellular cytotoxicity (ADCC) activity of immunoglobulin G monoclonal antibodies (mAbs). Here, we first prepared anti-HER2 mAbs having two core-fucosylated N-glycan chains with the single G2F, G1aF, G1bF, or G0F structure, together with those having two N-glycan chains with a single non-core-fucosylated corresponding structure for comparison, and determined their biological activities. Dissociation constants of mAbs with core-fucosylated N-glycans bound to recombinant Fcγ-receptor type IIIa variant were 10 times higher than those with the non-core-fucosylated N-glycans, regardless of core glycan structures. mAbs with the core-fucosylated N-glycans had markedly reduced ADCC activities, while those with the non-core-fucosylated N-glycans had high activities. These results indicate that the presence of a core-fucose residue in the N-glycan suppresses the binding to the Fc-receptor and the induction of ADCC of anti-HER2 mAbs. [ABSTRACT FROM AUTHOR]

Juvenile idiopathic arthritis (JIA) encompasses all forms of chronic idiopathic arthritis that arise before age 16. Previous studies have found JIA to be associated with lower Fc galactosylation of circulating IgG, but the overall spectrum of glycan changes and the net impact on IgG function are unknown. Using ultra performance liquid chromatography (UPLC), we compared IgG glycosylation in 54 subjects with recent-onset untreated JIA with 98 healthy pediatric controls, paired to biophysical profiling of affinity for 20 IgG receptors using a high-throughput multiplexed microsphere assay. Patients with JIA exhibited an increase in hypogalactosylated and hyposialylated IgG glycans, but no change in fucosylation or bisection, together with alteration in the spectrum of IgG ligand binding. Supervised machine learning demonstrated a robust capacity to discriminate JIA subjects from controls using either glycosylation or binding data. The binding signature was driven predominantly by enhanced affinity for Fc receptor like protein 5 (FcRL5), a noncanonical Fc receptor expressed on B cells. Affinity for FcRL5 correlated inversely with galactosylation and sialylation, a relationship confirmed through enzymatic manipulation. These results demonstrate the capacity of combined structural and biophysical IgG phenotyping to define the overall functional impact of IgG glycan changes and implicate FcRL5 as a potential cellular sensor of IgG glycosylation. [ABSTRACT FROM AUTHOR]

Core fucosylation, a posttranslational modification of N-glycans, modifies several growth factor receptors and impacts on their ligand binding affinity. Core-fucose-deficient mice generated by ablating the α1,6 fucosyltransferase enzyme, Fut8, exhibit severe pulmonary emphysema, partly due to impaired macrophage function, similar to aged Toll-like receptor 4 (Tlr4)-deficient mice. We therefore suspect that a lack of core fucose affects the TLR4-dependent signaling pathway. Indeed, upon lipopolysaccharide stimulation, Fut8-deficient mouse embryonic fibroblasts (MEFs) produced similar levels of interleukin-6 but markedly reduced levels of interferon-β (IFN-β) compared with wild-type MEFs. Lectin blot analysis of the TLR4 signaling complex revealed that core fucosylation was specifically found on CD14. Even though similar levels of TLR4/myeloid differentiation factor 2 (MD2) activation and dimerization were observed in Fut8-deficient cells after lipopolysaccharide stimulation, internalization of TLR4 and CD14 was significantly impaired. Given that internalized TLR4/MD2 induces IFN-β production, impaired IFN-β production in Fut8-deficient cells is ascribed to impaired TLR4/MD2 internalization. These data show for the first time that glycosylation critically regulates TLR4 signaling. [ABSTRACT FROM AUTHOR]

PMN‐expressed Lea represents a novel glycan target for modulation of PMN trafficking to mucosal tissues during inflammation. PMN‐expressed fucosylated glycans from the Lewis glycan family, including Lewis‐x (Lex) and sialyl Lewis‐x (sLex), have previously been implicated in the regulation of important PMN functions, including selectin‐mediated trafficking across vascular endothelium. Although glycans, such as Lex and sLex, which are based on the type 2 sequence (Galβ1‐4GlcNAc‐R), are abundant on PMNs, the presence of type 1 Galβ1‐3GlcNAc‐R glycans required for PMN expression of the closely related stereoisomer of Lex, termed Lewis‐A (Lea), has not, to our knowledge, been reported. Here, we show that Lea is abundantly expressed by human PMNs and functionally regulates PMN migration. Using mAbs whose precise epitopes were determined using glycan array technology, Lea function was probed using Lea‐selective mAbs and lectins, revealing increased PMN transmigration across model intestinal epithelia, which was independent of epithelial‐expressed Lea. Analyses of glycan synthetic machinery in PMNs revealed expression of β1‐3 galactosyltransferase and α1–4 fucosyltransferase, which are required for Lea synthesis. Specificity of functional effects observed after ligation of Lea was confirmed by failure of anti‐Lea mAbs to enhance migration using PMNs from individuals deficient in α1–4 fucosylation. These results demonstrate that Lea is expressed on human PMNs, and its specific engagement enhances PMN migration responses. We propose that PMN Lea represents a new target for modulating inflammation and regulating intestinal, innate immunity. [ABSTRACT FROM AUTHOR]

Fucosylated chondroitin sulfate (FCS) from sea cucumbers is composed of a chondroitin sulfate (CS) central core and branches of sulfated fucose. The structure of this complex glycosaminoglycan is usually investigated via nuclear magnetic resonance (NMR) analyses of the intact molecule, ergo through a top-down approach, which often yield spectra with intricate sets of signals. Here we employed a bottom-up approach to analyze the FCSs from the sea cucumbers Isostichopus badionotus and Ludwigothurea grisea from their basic constituents, viz. CS cores and sulfated fucose branches, obtained via systematic fragmentation through mild acid hydrolysis. Oligosaccharides derived from the central CS core were analyzed via NMR spectroscopy and the disaccharides produced using chondroitin sulfate lyase via SAX-HPLC. The CS cores from the two species were similar, showing only slight differences in the proportions of 4- or 6-monosulfated and 4,6-disulfated ß-D-GalNAc. Sulfated fucose units released from the FCSs were analyzed via NMR and ESI-HRMS spectroscopies. The fucose units from each species presented extensive qualitative differences, but quantitative assessments of these units were hindered, mostly because of their extensive desulfation during the hydrolysis. The bottom-up analysis performed here has proved useful to explore the structure of FCS through a sum-of-the-parts approach in a qualitative manner. We further demonstrate that under specific acidification conditions particular fucose branches can be removed preferentially from FCS. Preparation of derivatives enriched with particular fucose branches could be useful for studies on "structure vs. biological function" of FCS. [ABSTRACT FROM AUTHOR]

In this study, we examined the distribution of fucosylated glycans in mouse intestines using a lectin, BC2LCN (N-terminal domain of the lectin BC2L-C from Burkholderia cenocepacia), as a probe. BC2LCN is specific for glycans with a terminal Fucα1,2Galβ1,3-motif and it is a useful marker for discriminating the undifferentiated status of human induced/embryonic stem cells. Apparent BC2LCN reactivity was detected in the secretory granules of goblet cells in the ileum but not those in the colon. We also found distinctive reactivity in the crypt bottom, which is known as the stem cell zone, of the colon and the ileum. Other lectins for fucosylated glycans, including Ulex europaeus agglutinin-I, Pholiota squarrosa lectin and Aleuria aurantia lectin, did not exhibit similar reactivity in the crypt bottom. Remarkably, BC2LCN-positive epithelial cells could be labeled with a niche cell marker, c-Kit/CD117. Overall, our results indicate that intestinal niche cells express distinct fucosylated glycans recognized by BC2LCN. Increasing evidence suggests that the selfrenewal and proliferation of stem cells depend on specific signals derived from niche cells. Our results highlight novel molecular properties of intestinal niche cells in terms of their glycosylation, which may help to understand the regulation of intestinal stem cells. The distinct expression of glycans may reflect the functional roles of niche cells. BC2LCN is a valuable tool for investigating the functional significance of protein glycosylation in stem cell regulation. [ABSTRACT FROM AUTHOR]

The functions of cell surface proteins, such as growth factor receptors and virus/bacteria-entry receptors, can be dynamically regulated by oligosaccharide modifications. In the present study, we investigated the involvement of glycosylation in hepatitis B virus (HBV) entry into hepatoma cells. Infection of oligosaccharide-remodeling hepatoma cells with a pseudo virus of HBV, bionanocapsule (BNC), was evaluated by flow cytometry and confocal microscopy. Among various experiments using several hepatoma cells, marked difference was observed between Huh6 cells and HB611 cells, which were established by HBV gene transfection into hepatoma cells. Comprehensive oligosaccharide analysis showed dramatic increases of core fucosylation in HB611 cells, compared with Huh6 cells. Knock down of fucosyltransferase 8 (FUT8) reduced BNC entry into HB611 cells. In contrast, overexpression of FUT8 in Huh6 cells increased BNC entry. Although expression of sodium taurocholate cotransporting polypeptide (NTCP), which is one of HBV receptors was very similar between Huh6 and HB611 cells, proteins coprecipitated with NTCP were dependent on levels of core-fucosylation, suggesting that core-fucosylation regulates BNC entry into hepatoma cells. Our findings demonstrate that core-fucosylation is an important glycosylation for HBV infection of hepatoma cells through HBV-receptor-mediated endocytosis. Down-regulation of core-fucosylation may be a novel target for HBV therapy. [ABSTRACT FROM AUTHOR]

The plant cell wall is a complex and dynamic network made mostly of cellulose, hemicelluloses, and pectins. Xyloglucan, the major hemicellulosic component in Arabidopsis thaliana , is biosynthesized in the Golgi apparatus by a series of glycan synthases and glycosyltransferases before export to the wall. A better understanding of the xyloglucan biosynthetic machinery will give clues toward engineering plants with improved wall properties or designing novel xyloglucan-based biomaterials. The xyloglucan-specific α2-fucosyltransferase FUT1 catalyzes the transfer of fucose from GDP-fucose to terminal galactosyl residues on xyloglucan side chains. Here, we present crystal structures of Arabidopsis FUT1 in its apoform and in a ternary complex with GDP and a xylo-oligosaccharide acceptor (named XLLG). Although FUT1 is clearly a member of the large GT-B fold family, like other fucosyltransferases of known structures, it contains a variant of the GT-B fold. In particular, it includes an extra C-terminal region that is part of the acceptor binding site. Our crystal structures support previous findings that FUT1 behaves as a functional dimer. Mutational studies and structure comparison with other fucosyltransferases suggest that FUT1 uses a SN2-like reaction mechanism similar to that of protein- O -fucosyltransferase 2. Thus, our results provide new insights into the mechanism of xyloglucan fucosylation in the Golgi. [ABSTRACT FROM AUTHOR]

Human mesenchymal stem cells (MSCs) hold great promise in cellular therapeutics for skeletal diseases but lack expression of E-selectin ligands that direct homing of blood-borne cells to bone marrow. Previously, we described a method to engineer E-selectin ligands on the MSC surface by exofucosylating cells with fucosyltransferase VI (FTVI) and its donor sugar, GDP-Fucose, enforcing transient surface expression of the potent E-selectin ligand HCELL with resultant enhanced osteotropism of intravenously administered cells. Here, we sought to determine whether E-selectin ligands created via FTVI-exofucosylation are distinct in identity and function to those created by FTVI expressed intracellularly. To this end, we introduced synthetic modified mRNA encoding FTVI ( FUT6-modRNA) into human MSCs. FTVI-exofucosylation (i.e., extracellular fucosylation) and FUT6-modRNA transfection (i.e., intracellular fucosylation) produced similar peak increases in cell surface E-selectin ligand levels, and shear-based functional assays showed comparable increases in tethering/rolling on human endothelial cells expressing E-selectin. However, biochemical analyses revealed that intracellular fucosylation induced expression of both intracellular and cell surface E-selectin ligands and also induced a more sustained expression of E-selectin ligands compared to extracellular fucosylation. Notably, live imaging studies to assess homing of human MSC to mouse calvarium revealed more osteotropism following intravenous administration of intracellularly-fucosylated cells compared to extracellularly-fucosylated cells. This study represents the first direct analysis of E-selectin ligand expression programmed on human MSCs by FTVI-mediated intracellular versus extracellular fucosylation. The observed differential biologic effects of FTVI activity in these two contexts may yield new strategies for improving the efficacy of human MSCs in clinical applications. S tem C ells 2016;34:2501-2511 [ABSTRACT FROM AUTHOR]

Background: Fucose is utilized for the modification of different molecules involved in blood group determination, immunological reactions, and signal transduction pathways. We have recently reported that enhanced activity of the fucosyltransferase 3 and/or 6 promoted TGF-ß-mediated epithelial mesenchymal transition and was associated with increased metastatic potential of colorectal cancer (CRC), suggesting that fucose is required by CRC cells. With this in mind, we examined requirement of L-fucose in CRC cells and developed fucose-bound nanoparticles as vehicles for delivery of anticancer drugs specific to CRC.Methods: In this study, we first examined the expression of fucosylated proteins in 50 cases of CRC by immunochistochemical staining with biotinylated Aleuria aurantia lectin (AAL). Then we carried out an L-fucose uptake assay using three CRC cell lines. Finally, we developed fucose-bound nanoparticles as vehicles for the delivery of an anticancer drug, SN38, and examined tumor growth inhibition in mouse xenograft model (n = 6 mice per group). All statistical tests were two-sided.Results: We found a statistically significant relationship between vascular invasion, clinical stage, and intensity score of AAL staining (P ≤ .02). L-fucose uptake assay revealed that L-fucose incorporation, as well as fucosylated protein release, was high in cells rich in fucosylated proteins. L-fucose-bound liposomes effectively delivered Cy5.5 into CRC cells. The excess of L-fucose decreased the efficiency of Cy5.5 uptake through L-fucose-bound liposomes, suggesting an L-fucose receptor dependency. Intravenously injected, L-fucose-bound liposomes carrying SN38 were successfully delivered to CRC cells, mediating efficient tumor growth inhibition (relative tumor growth ratio: no treatment group [NT], 8.29 ± 3.09; SN38-treated group [SN38], 3.53 ± 1.47; liposome-carrying, SN38-treated group [F0], 3.1 ± 1.39; L-fucose-bound, liposome-carrying, SN38-treated group [F50], 0.94 ± 0.89; F50 vs NT, P = .003; F50 vs SN38, P = .02, F50 vs F0, P = .04), as well as prolonging survival of mouse xenograft models (log-rank test, P < .001).Conclusions: Thus, fucose-bound liposomes carrying anticancer drugs provide a new strategy for the treatment of CRC patients. [ABSTRACT FROM AUTHOR]

Three novel bacterial a-L-fucosidases, which cleave terminal fucosyl residues from glycoconjugates are reported in this work. Originating from the recently discovered bacterium Emticicia oligotrophica, recombinant fucosidase isoforms designated as Eo0918, Eo3066 and Eo3812 were shown to have the highest activity between pH 6.0 and 7.0 and temperature optima between 30 and 45°C. All enzymes catalyzed the hydrolysis of the model substrate pNP-a-L-fucose and revealed significantly different regiospecificities towards fucose-containing oligosaccharides: Eo0918 liberated exclusively a1,6-linked fucose and Eo3812 released only a1,3-fucosyl residues, whereas Eo3066 showed broader substrate promiscuity. The enzymatic activity of Eo0918 and Eo3812 increased upon the addition of Ca2+, Mn2+ and Zn2+ ions, whereas the activity of Eo3066 was significantly decreased in the presence of these metal ions. In addition, Eo0918 also catalyzed the transfer of fucose from pNP-a-L-fucose to the 7-hydroxyl group of 4-methylumbelliferone with up to 15%transglycosylation yield. Facile recombinant expression in E. coli, distinct substrate specificities and the transglycosylation ability of Eo0918 presented herein make these newly discovered fucosidases valuable candidates for bioanalytical and biotechnological applications. [ABSTRACT FROM AUTHOR]

Protein glycosylation is one of the most significant post-translation modifications and plays a critical role in various biological functions. Haptoglobin (Hp) is one of the acute-phase response proteins secreted by liver. Its glycosylation could be analyzed by many analytical techniques qualitatively and quantitatively. The glycosylation alterations of Hp are reported to be associated with different kinds of diseases. The main glycosylation alterations of Hp in cancer appear to be the presence of aberrantly fucosylated and sialylated structures as well as increased branching. In this review we provided a brief overview of Hp structure and biological function, discussed its glycosylation alterations in different cancers, and described the existing technologies for analyzing glycosylation site and glycan of Hp. Given the importance of Hp glycosylation, its unknown and unclear biological complexity and significance, Hp glycosylation has become a major target in cancer research. Development of sensitive and specific detection of Hp glycosylation including large-scale validation may provide significant steps forward to its clinical application. [ABSTRACT FROM AUTHOR]

A fucosylated chondroitin sulfate (FCS) was isolated from the body wall of Pacific sea cucumber Cucumaria japonica by extraction in the presence of papain followed by Cetavlon precipitation and anion-exchange chromatography. FCS was shown to contain D-GalNAc, D-GlcA, L-Fuc and sulfate in molar proportions of about 1:1:1:4.5. Structure of FCS was elucidated using NMR spectroscopy and methylation analysis of the native polysaccharide and products of its desulfation and carboxyl reduction. The polysaccharide was shown to contain a typical chondroitin core →3)-β-D-GalNAc- (1→4)-β-D-GlcA-(1→. Sulfate groups in this core occupy O-4 and the majority of O-6 of GalNAc. Fucosyl branches are represented by 3,4- and 2,4-disulfated units in a ratio of 4:1 and are linked to O-3 of GlcA. In addition, ~33% of GlcA are 3-O-sulfated, and hence, the presence of short fucooligosaccharide chains side by side with monofucosyl branches cannot be excluded. FCS was shown to inhibit platelets aggregation in vitro mediated by collagen and ristocetin, but not adenosine diphosphate, and demonstrated significant anticoagulant activity, which is connected with its ability to enhance inhibition of thrombin and factor Xa by antithrombin III, as well as to influence vonWillebrand factor activity. The latest property significantly distinguished FCS from low-molecular-weight heparin. [ABSTRACT FROM AUTHOR]

Human milk oligosaccharides (HMOs) are recognized as benefiting breast-fed infants in multiple ways. As a result, there is growing interest in the synthesis of HMOs mimicking their natural diversity. Most HMOs are fucosylated oligosaccharides. α-L-Fucosidases catalyze the hydrolysis of α-L-fucose from the non-reducing end of a glucan. They fall into the glycoside hydrolase GH29 and GH95 families. The GH29 family fucosidases display a classic retaining mechanism and are good candidates for transfucosidase activity. We recently demonstrated that the α-L-fucosidase from Thermotoga maritima (TmaFuc) from the GH29 family can be evolved into an efficient transfucosidase by directed evolution (Osanjo et al. 2007). In this work, we developed semi-rational approaches to design an α-Ltransfucosidase starting with the α-L-fucosidase from commensal bacteria Bifidobacterium longum subsp. infantis (BiAfcB, Blon_2336). Efficient fucosylation was obtained with enzyme mutants (L321P-BiAfcB and F34I/L321P-BiAfcB) enabling in vitro synthesis of lactodifucotetraose, lacto-N-fucopentaose II, lacto-N-fucopentaose III and lacto-N-difucohexaose I. The enzymes also generated more complex HMOs like fucosylated para-lacto-N-neohexaose (F-p-LNnH) and mono- or difucosylated lacto-N-neohexaose (F-LNnH-I, F-LNnH-II and DF-LNnH). It is worth noting that mutation at these two positions did not result in a strong decrease in the overall activity of the enzyme, which makes these variants interesting candidates for large-scale transfucosylation reactions. For the first time, this work provides an efficient enzymatic method to synthesize the majority of fucosylated HMOs. [ABSTRACT FROM AUTHOR]

E-cadherin is often dysregulated in aggressive lung cancer, the mechanism of which cannot always be explained at the level of transcription. In 66 patients with lung cancer, immunohistochemical staining demonstrated that co-localization of E-cadherin and core fucose by Lens culinaris agglutinin was significantly less extensive in tumor than in nontumor tissue. Through gain and loss of fucosylation experiments in the giant lung carcinoma cell lines 95C and 95D, our results revealed that E-cadherin core fucosylation in 95C cells overexpressing α-1, 6-fucosyltransferase (Fut8) inhibited Fut8-95C cell migration, whereas knockdown of Fut8 in 95D cells enhanced migration of shortinterfering RNA-targeting Fut8 (siFut8)-95D cells. The level of active Src (phosphorylated Src [Y416]) was significantly reduced in Fut8-95C cells, but elevated in siFut8-95D cells. In protein complexes immunoprecipitated from Fut8-95C cell lysates with anti-E-cadherin, less phosphorylated Src (Y416) and more β-catenin were observed, but immunoprecipitates from siFut8-95D cells, containing less core fucosylated E-cadherin, contained an elevated level of phospho-Src Y416. In Fut8-95C cells, phosphorylation of Akt (Y315, Y326) and GSK-3β (S9) was significantly reduced, but β-catenin (S37) phosphorylation was enhanced. Expression of N-cadherin and Snail1 was also reduced in Fut8-95C cells, but significantly increased in siFut8-95D cells. Intriguingly, when Src kinase activity was inhibited by treatment of cells with PP2 and SU6656, regulation of N-cadherin, Snail1 and cell migration by E-cadherin core fucosylation was abrogated in both Fut8-95C and siFut8-95D cells. Therefore, posttranslational modification of E-cadherin by less core fucosylation recruited and activated Src, and induced an epithelial-mesenchymal transition-like process in lung cancer cells. [ABSTRACT FROM AUTHOR]

Fucosylated chondroitin sulfate (FCS) is a glycosaminoglycan found in sea cucumbers. It has a backbone like that of mammalian chondroitin sulfate (4-β-D-GlcA-1→3-β-D-GalNAc-1)n but substituted at the 3rd position of the β-D-glururonic acid residues with a-fucose branches. The structure of these branches varies among FCSs extracted from different species of sea cucumbers, as revealed by solution NMR spectroscopy. Some species (Isostichopus badionotus and Patalus mollis) contain branches formed by single a-fucose residues but with variable sulfation patterns (2,4-, 3,4- and 4-sulfation). FCS from Ludwigothurea grisea is distinguished because it contains preponderant branches formed by disaccharide units containing non-sulfated and 3-sulfated a-fucose units at the reducing and non-reducing ends, respectively. Despite the structural variability on their a-fucose branches, these FCSs have similar anticoagulant action on assays using purified reagents. They have serpin-dependent and serpin-independent effects. Pharmacological assays using experimental animals showed that the three types of FCSs have similar antithrombotic effect and bleeding tendency. They also activate factor XII on the same range of concentration. Based on these observations, we proposed that only few sulfated a-fucose branches along the FCS chain are enough to assure the binding of this glycosaminoglycan to proteins of the coagulation system. Substitution with additional sulfated a-fucose does not increase further the activity. Overall, the use of FCSs with marked variability on their branches of a-fucose allowed us to establish correlations between structures vs biological effects of these glycosaminoglycans on a more refined basis. It opens new avenues for therapeutic intervention using FCSs. [ABSTRACT FROM AUTHOR]

STUDY QUESTION: Could seminal plasma clusterin play a role in the uptake of stress-damaged proteins by dendritic cells? SUMMARY ANSWER: Seminal plasma clusterin, but not serum clusterin, promotes the uptake of stress-damaged proteins by dendritic cells via DC-SIGN. WHAT IS KNOWN ALREADY: Clusterin is one of the major extracellular chaperones. It interacts with a variety of stressed proteins to prevent their aggregation, guiding them for receptor-mediated endocytosis and intracellular degradation. The concentration of clusterin in semen is almost 20-fold higher than that found in serum, raising the question about the role of seminal plasma clusterin in reproduction. No previous studies have analyzed whether seminal plasma clusterin has chaperone activity. We have previously shown that seminal plasma clusterin, but not serum clusterin, expresses an extreme abundance of fucosylated glycans. These motifs enable seminal plasma clusterin to bind DC-SIGN with very high affinity. STUDY DESIGN, SIZE, DURATION: In vitro experiments were performed to evaluate the ability of seminal plasma clusterin to inhibit the precipitation of stressed proteins, promoting their uptake by dendritic cells via DC-SIGN (a C-typelectin receptor selectively expressed on dendritic cells (DC)). Moreover, the ability of seminal plasma clusterin to modulate the phenotype and function of DCs was also assessed. PARTICIPANTS/MATERIALS, SETTING, METHODS: Clusterin was purified from human semen and human serum. Catalase, bovine serum albumin, glutathione S-transferase, and normal human serum were stressed and the ability of seminal plasma clusterin to prevent the precipitation of these proteins, guiding them to DC-SIGN expressed by DCs, was evaluated using a fluorescence-activated cell sorter (FACS). Endocytosis of stressed proteins was analyzed by confocal microscopy and the ability of seminal plasma clusterin-treated DCs to stimulate the proliferation of CD25 + FOXP3 + CD4+ T cells was also evaluated by FACS. MAIN RESULTS AND THE ROLE OF CHANCE: Seminal plasma clusterin interacts with stressed proteins, inhibits their aggregation (P < 0.01) and efficiently targets them to dendritic cells via DC-SIGN (P < 0.01). DCs efficiently endocytosed clusterin-client complexes and sorted them to degradative compartments involved in antigen processing and presentation. Moreover, we also found that the interaction of seminal plasma clusterin with DC-SIGN did not change the phenotype of DCs, but stimulates their ability to induce the expansion of CD25 + FOXP3 + CD4+ T lymphocytes (P < 0.05 versus control). LIMITATIONS, REASONS FOR CAUTION: All the experiments were performed in vitro; hence the relevance of our observations should be validated in vivo. WIDER IMPLICATIONS OF THE FINDINGS: Our results suggest that by inducing the endocytosis of stress-damaged proteins by DCs via DC-SIGN, seminal plasma clusterin might promote a tolerogenic response to male antigens, thereby contributing to female tolerance to seminal antigens. STUDY FUNDING/COMPETING INTEREST(S): The present research was supported by the Consejo Nacional de Investigaciones Científicas y Técnicas, the Buenos Aires University School of Medicine, and the Agencia Nacional de Promoción Científica y Tecnológica (Argentina). The authors have no conflicts of interest to declare. [ABSTRACT FROM AUTHOR]

The predominant structure of the hemicellulose xyloglucan (XyG) found in the cell walls of dicots is a fucogalactoXyG with an XXXG core motif, whereas in the Poaceae (grasses and cereals), the structure of XyG is less xylosylated (XXGGn core motif) and lacks fucosyl residues. However, specialized tissues of rice (Oryza sativa) also contain fucogalactoXyG. Orthologous genes of the fucogalactoXyG biosynthetic machinery of Arabidopsis (Arabidopsis thaliana) are present in the rice genome. Expression of these rice genes, including fucosyl-, galactosyl-, and acetyltransferases, in the corresponding Arabidopsis mutants confirmed their activity and substrate specificity, indicating that plants in the Poaceae family have the ability to synthesize fucogalactoXyG in vivo. The data presented here provide support for a functional conservation of XyG structure in higher plants. [ABSTRACT FROM AUTHOR]

We determined the specificity of BTL, a lectin from the red marine alga Bryothamnion triquetrum, toward fucosylated oligosaccharides. BTL showed a strict specificity for the core α1,6-fucosylation, which is an important marker for cancerogenesis and quality control of therapeutical antibodies. The double fucosylation α1,6 and α1,3 was also recognized, but the binding was totally abolished in the sole presence of the α1,3-fucosylation. A more detailed analysis of the specificity of BTL showed a preference for bi- and tri-antennary nonbisected N-glycans. Sialylation or fucosylation at the nonreducing end of N-glycans did not affect the recognition by the lectin. BTL displayed a strong affinity for a core α1,6-fucosylated octasaccharide with a Kd of 12 μM by titration microcalorimetry. The structural characterization of the interaction between BTL and the octasaccharide was obtained by STD-NMR. It demonstrated an extended epitope for recognition that includes the fucose residue, the distal GlcNAc and one mannose residue. Recombinant rBTL was obtained in Escherichia coli and characterized. Its binding properties for carbohydrates were studied using hemagglutination tests and glycan array analysis. rBTL was able to agglutinate rabbit erythrocytes with strong hemagglutination activity only after treatment with papain and trypsin, indicating that its ligands were not directly accessible at the cell surface. The hemagglutinating properties of rBTL confirm the correct folding and functional state of the protein. The results show BTL as a potent candidate for cancer diagnosis and as a reagent for the preparation and quality control of antibodies lacking core α1,6-fucosylated N-glycans. [ABSTRACT FROM AUTHOR]

N-Glycans from the nematode Haemonchus contortus (barber pole worm), a parasite of sheep and cattle, were the first to be described to possess up to three fucose residues associated with the N,N'-diacetylchitobiosyl core, two being on the reducing-terminal proximal GlcNAc and one on the distal core GlcNAc residue. The assumption was that truncated glycans from this organism with three hexose residues have the composition Man3GlcNAc2Fuc1-3. In this study, we have performed HPLC and MALDI-TOF MS/MS in combination with selected digestions of N-glycans from Haemonchus. A dominant trifucosylated Hex3HexNAc2Fuc3 glycan was modified not only with α1,6-fucose but also with a proximal core α1,3-fucose and a galactosylated distal α1,3-fucose; thereby, only two of the hexose residues were mannose. Other N-glycans displayed galactosylation of the core α1,6-fucose, antennal fucosylation or modification with phosphorylcholine. Thus, the N-glycans of Haemonchus contain a number of potentially immunogenic glycan epitopes also found in other parasites and our proposed structures are in line with the previously defined specificity of nematode glycosyltransferases as we show that distal fucosylation and the presence of an α1,6-mannose are apparently mutually exclusive. These data are thereby of importance for engineering cell lines capable of mimicking Haemonchus-type N-glycans in the preparation of recombinant proteins as vaccine candidates. [ABSTRACT FROM AUTHOR]

Background and Aims In flowering plants, fertilization relies on the delivery of the sperm cells carried by the pollen tube to the ovule. During the tip growth of the pollen tube, proper assembly of the cell wall polymers is required to maintain the mechanical properties of the cell wall. Xyloglucan (XyG) is a cell wall polymer known for maintaining the wall integrity and thus allowing cell expansion. In most angiosperms, the XyG of somatic cells is fucosylated, except in the Asterid clade (including the Solanaceae), where the fucosyl residues are replaced by arabinose, presumably due to an adaptive and/or selective diversification. However, it has been shown recently that XyG of Nicotiana alata pollen tubes is mostly fucosylated. The objective of the present work was to determine whether such structural differences between somatic and gametophytic cells are a common feature of Nicotiana and Solanum (more precisely tomato) genera.Methods XyGs of pollen tubes of domesticated (Solanum lycopersicum var. cerasiforme and var. Saint-Pierre) and wild (S. pimpinellifolium and S. peruvianum) tomatoes and tobacco (Nicotiana tabacum) were analysed by immunolabelling, oligosaccharide mass profiling and GC-MS analyses.Key Results Pollen tubes from all the species were labelled with the mAb CCRC-M1, a monoclonal antibody that recognizes epitopes associated with fucosylated XyG motifs. Analyses of the cell wall did not highlight major structural differences between previously studied N. alata and N. tabacum XyG. In contrast, XyG of tomato pollen tubes contained fucosylated and arabinosylated motifs. The highest levels of fucosylated XyG were found in pollen tubes from the wild species.Conclusions The results clearly indicate that the male gametophyte (pollen tube) and the sporophyte have structurally different XyG. This suggests that fucosylated XyG may have an important role in the tip growth of pollen tubes, and that they must have a specific set of functional XyG fucosyltransferases, which are yet to be characterized. [ABSTRACT FROM PUBLISHER]

Glycosylation is an important attribute of baculovirus-insect cell expression systems, but some insect cell lines produce core α1,3-fucosylated N-glycans, which are highly immunogenic and render recombinant glycoproteins unsuitable for human use. To address this problem, we exploited a bacterial enzyme, guanosine-5′-diphospho (GDP)-4-dehydro-6-deoxy-d-mannose reductase (Rmd), which consumes the GDP-l-fucose precursor. We expected this enzyme to block glycoprotein fucosylation by blocking the production of GDP-l-fucose, the donor substrate required for this process. Initially, we engineered two different insect cell lines to constitutively express Rmd and isolated subclones with fucosylation-negative phenotypes. However, we found the fucosylation-negative phenotypes induced by Rmd expression were unstable, indicating that this host cell engineering approach is ineffective in insect systems. Thus, we constructed a baculovirus vector designed to express Rmd immediately after infection and facilitate the insertion of genes encoding any glycoprotein of interest for expression later after infection. We used this vector to produce a daughter encoding rituximab and found, in contrast to an Rmd-negative control, that insect cells infected with this virus produced a nonfucosylated form of this therapeutic antibody. These results indicate that our Rmd+ baculoviral vector can be used to solve the immunogenic core α1,3-fucosylation problem associated with the baculovirus-insect cell system. In conjunction with existing glycoengineered insect cell lines, this vector extends the utility of the baculovirus-insect cell system to include therapeutic glycoprotein production. This new vector also extends the utility of the baculovirus-insect cell system to include the production of recombinant antibodies with enhanced effector functions, due to its ability to block core α1,6-fucosylation. [ABSTRACT FROM AUTHOR]

The conserved oligomeric Golgi complex (COG) is a hetero-octomeric peripheral membrane protein required for retrograde vesicular transport and glycoconjugate biosynthesis within the Golgi. Mutations in subunits 1, 4, 5, 6, 7 and 8 are the basis for a rare inheritable human disease termed congenital disorders of glycosylation type-II. Defects to COG complex function result in aberrant glycosylation, protein trafficking and Golgi structure. The cellular function of the COG complex and its role in protein glycosylation are not completely understood. In this study, we report the first detailed structural analysis of N-glycans from a COG complex-deficient organism. We employed sequential ion trap mass spectrometry of permethylated N-glycans to demonstrate that the COG complex is essential for the formation of fucose-rich N-glycans, specifically antennae fucosylated structures in Caenorhabditis elegans. Our results support the supposition that disruption to the COG complex interferes with normal protein glycosylation in the medial and/or trans-Golgi. [ABSTRACT FROM PUBLISHER]

Ribosome display was applied to the Fc region of human immunoglobulin G (IgG1) to select for improved binding to human FcγRIIIa, the receptor expressed on human natural killer cells that mediates antibody-dependent cellular cytotoxicity (ADCC). A library of human Fcγ1 variants was generated using error-prone polymerase chain reaction, and subjected to multiple rounds of ribosome display selection against progressively decreasing concentrations of soluble human FcγRIIIa, to enrich for improved binders. Radioimmunoassay and alphascreen analyses of the aglycosylated IgG-Fc output revealed variants with improved binding to FcγRIIIa relative to wild-type IgG-Fc. Subsequent expression in human (HEK-EBNA) cells generated glycosylated IgGs with modified activity in ADCC assays. One particular variant, 125_B01 triggered enhanced ADCC (EC50 up to four-fold reduced with increased maximal lysis) relative to wild-type antibody, having more equal levels of ADCC for each allotype (V158/F158) of FcγRIIIa. Deconvolution of individual replacements within the variant showed that improved function arose from the Phe243Leu replacement within the CH2 domain, rather than the CH3 domain replacements Thr393Ala or His433Pro. Surprisingly, the oligosaccharide profiles of 125_B01 indicated more oligosaccharide chains lacking fucose, or with bisecting N-acetylglucosamine relative to wild-type IgG1, which correlates with improved function and the replacement Phe243Leu that is a carbohydrate contact residue within the CH2 domain. [ABSTRACT FROM AUTHOR]

FUT8, a eukaryotic α1,6-fucosyltransferase, catalyzes the transfer of a fucosyl residue from guanine nucleotide diphosphate-β-l-fucose to the innermost GlcNAc of an asparagine-linked oligosaccharide (N-glycan). The catalytic domain of FUT8 is structurally similar to that of NodZ, a bacterial α1,6-fucosyltransferase, which acts on a chitooligosaccharide in the synthesis of Nod factor. While the substrate specificities for the nucleotide sugar and the N-glycan have been determined, it is not known whether FUT8 is able to fucosylate other sugar chains such as chitooligosaccharides. The present study was conducted to investigate the action of FUT8 on chitooligosaccharides that are not generally thought to be a substrate in mammals, and the results indicate that FUT8 is able to fucosylate such structures in a manner comparable to NodZ. Surprisingly, structural analyses of the fucosylated products by high performance liquid chromatography, mass spectrometry and nuclear magnetic resonance indicated that FUT8 does not utilize the reducing terminal GlcNAc for fucose transfer but shows a preference for the third GlcNAc residue from the nonreducing terminus of the acceptor. These findings suggest that FUT8 catalyzes the fucosylation of chitooligosaccharide analogous to NodZ, but that a nonreducing terminal chitotriose structure is required for the reaction. The substrate recognition by which FUT8 selects the position to fucosylate might be distinct from that for NodZ and could be due to structural factor requirements which are inherent in FUT8. [ABSTRACT FROM PUBLISHER]

The article presents a study which examines the expression of fucosylated glycosphingolipid species in rat prostate tissue. The method of the study includes lection blot analysis, preparation of subcellular fractions, lectin blotting, and enzyme digestion. Results from the analysis of lectin blotting shows that prostatic focusylated glycoconjugates (PFGs) is expressed in the ventral prostates of the rat and PFGs expression is associated with some androgen-related processes in the tissue.