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Saccharides form one of the major constituents of biological macromolecules in living organisms. Many biological processes including protein folding, stability, immune response and receptor activation are regulated by glycosylation. In this work, we optimized a capillary electrophoresis method with capacitively coupled contactless conductivity detection for the separation of eight monosaccharides commonly found in glycoproteins, namely D-glucose, D-galactose, D-mannose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, D-fucose, N-acetylneuraminic acid, and D-xylose. A highly alkaline solution of 50 mM sodium hydroxide, 22.5 mM disodium phosphate, and 0.2 mM CTAB (pH 12.4) was used as a background electrolyte in a 10 µm id capillary. To achieve baseline separation of all analytes, a counter-directional pressure of -270 kPa was applied during the separation. The limits of detection of our method were below 7 µg/ml (i.e., 1.5 pg or 1 mg/g protein) and the limits of quantification were below 22 µg/ml (i.e., 5 pg or 3 mg/g protein). As a proof of concept of our methodology, we performed an analysis of monosaccharides released from fetuin glycoprotein by acid hydrolysis. The results show that, when combined with an appropriate pre-concentration technique, the developed method can be used as a monosaccharide profiling tool in glycoproteomics and complement the routinely used LC-MS/MS analysis.

Mucin glycoproteins are essential components of the mucosal barrier, which protects the host from pathogens. Throughout evolution, bacteria have developed strategies to modulate and penetrate this barrier, and cause virulence by interacting with mucin O-glycans at the epithelial cell-surface. O-fucosylated glycan epitopes on mucins are key ligands of many bacterial lectins. Here, a chemoenzymatic synthesis strategy is described to prepare a library of fucosylated mucin core glycopeptides to enable studies of mucin-interacting and fucose-binding bacterial lectins. Glycan cores with biologically important Lewis and H-antigens were prepared decorating the peptide backbone at different sites and densities. The fucosylated mucin glycopeptides were applied in microarray binding studies to explore the importance of glycan core and peptide backbone presentation of these antigens in binding interactions with the P. aeruginosa lectin LecB and the C. difficile toxin A.

l-Fucose, as a monosaccharide in nature, plays a crucial role in bacteria colonization. Escherichia coli (E. coli), as a common microorganism in environment, utilize bacterial flagellar motor to drive the rotation of flagella, which is regulated by chemotactic signal transduction signals. Yet the effect of l-fucose to bacterial motility remains unclear. The effect of l-fucose on the swimming motility of bacteria was investigated from the level of single flagellar motor to individual cell and cell population by employing a bead assay, a high-throughput 2D tracking assay and a high-throughput dark-field flicker microscopy. The results showed that the swimming motility of the bacteria cultured with l-fucose was decreased, while the tumble frequency increased. Furthermore, the behavioral alterations of bacteria affected by l-fucose were directly reveled by measuring the cell distribution of bacteria swimming near surfaces and bacterial surface adhesion, suggesting that l-fucose promotes bacterial surface aggregation and surface adhesion. The effect of l-fucose on bacterial swimming motility characterized in this study are consistent with the key role that l-fucose plays in bacterial colonization.

BACKGROUND The high yield of ultrasonic extraction has been widely studied. However, the effects of ultrasound on the properties of products has generally been ignored. In this study, the structural characteristics, rheological properties, and thermal stability of Flammulina velutipes polysaccharides (FVPs) under different ultrasonic power (200, 600, 1000 W) and time (10, 20, 30 min) were investigated to explore the effects of ultrasonic extraction on FVPs and the structure-physicochemical properties relationship. The ultrasonic intensity at the corresponding rated power was also measured. RESULTS The results showed that the molecular weight, particle size, and zeta potential of FVPs decreased as the ultrasonic intensity or time increased. The galactose, mannose, and fucose contents were increased, but the glucose content was decreased by ultrasonic extraction. Viscosity and weak gel strength were positively correlated with molecular weight. Thermal degradation enthalpy was positively correlated with the galactose and fucose contents. CONCLUSIONS Ultrasound reduced the viscosity and gel strength of FVPs by breaking the polysaccharide chain and improving the galactose and fucose contents, which improved the thermal stability of FVPs. This work provides a theoretical basis for the development of FVP foods with a clear structure-function relationship, which makes it possible to directionally produce FVPs by adjusting ultrasonic parameters during extraction. © 2021 Society of Chemical Industry.

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

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

O-linked modification of nuclear and cytosolic proteins with monosaccharides is essential in all eukaryotes. While many aspects of this post-translational modification are highly conserved, there are striking differences between plants and the animal kingdom. In animals, dynamic cycling of O-GlcNAc is established by two essential single copy enzymes, the O-GlcNAc transferase OGT and O-GlcNAc hydrolase OGA. In contrast, plants balance O-GlcNAc with O-fucose modifications, catalyzed by the OGT SECRET AGENT (SEC) and the protein O-fucosyltransferase (POFUT) SPINDLY (SPY). However, specific glycoside hydrolases for either of the two modifications have not yet been identified. Nucleocytoplasmic O-glycosylation is still not very well understood in plants, even though a high number of proteins were found to be affected. One important open question is how specificity is established in a system where only two enzymes modify hundreds of proteins. Here, we discuss the possibility that O-GlcNAc- and O-fucose-binding proteins could introduce an additional flexible layer of regulation in O-glycosylation-mediated signaling pathways, with the potential of integrating internal or external signals.

Amoebic gill disease (AGD) is caused by the marine amoeba Neoparamoeba perurans, a facultative parasite. Despite the significant impact this disease has on production of Atlantic salmon worldwide, the mechanisms involved in host-parasite interaction remains unknown. Excessive gill mucus secretion is reported as a host defence mechanism to prevent microbial colonization in the gill epithelium. Despite this response, N. perurans still attaches and proliferates. The present study aimed to investigate the interaction between N. perurans and mucin, the most abundant component in mucus. An in vitro adhesion assay using bovine submaxillary mucin (BSM) demonstrated that amoeba binding to mucin-coated substrate was significantly higher than to the BSA control. This binding interaction is likely glycan-mediated as pre-incubation with galactose, galactosamine, N-acetylgalactosamine and fucose reduced mucin adhesion to control levels. The ability of N. perurans to secrete proteases that target mucin was also investigated. Protease activity was detected in the amoeba culture media in the presence of BSM, but not when protease inhibitor was added. Mucin degradation was visually assessed on protein gels. This study provides preliminary evidence that N. perurans has developed mechanisms to interact with and evade mucus by binding to mucin glycan receptors and secreting proteases with mucolytic activity.

It is known that core-type fucosylation is higher in prostate cancer cells than in other cancer cell types and is associated with high-risk prostate cancer. Here, we developed an automated microcapillary electrophoresis-based immunoassay system for measuring serum core-type fucosylated prostate-specific antigen (PSA) and evaluated whether the serum fucosylated PSA index (FPI) can detect high-risk prostate cancer. Core-type fucosylated-free PSA was measured by our automated microcapillary electrophoresis-based immunoassay system with Pholiota squarrosa lectin. The FPI was calculated from total PSA and the percentage of fucosylated-free PSA. The optimum model to predict Gleason grade (GG) ≥2 was constructed by multivariate logistic regression analysis. Discrimination was assessed by determining the area under the receiver operator characteristic curve (AUC). The study included 252 men who underwent prostate needle biopsy due to elevated serum PSA levels (4-20 ng/mL), including 138 with GG ≥2. A higher FPI was significantly associated with GG (P

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

The L-fucose-specific lectin from Aspergillus niger (ANL), isolated from the corneal smears of a keratitis patient was reported earlier. Here, we examined the interaction of ANL with human hepatocellular and colon cancer cells, evaluated its anti-cancer activity and diagnostic potential to detect aberrantly glycosylated tumour-associated serum glycoproteins such as alpha-fetoprotein (AFP). We observed that ANL strongly bound to both HepG2 and HT-29 cell-lines and this interaction was effectively blocked with L-fucose and mucin in a dose and time-dependent manner with an IC50 of 1.25 and 5 μg/mL for HepG2 and HT-29 cells respectively at 48 hours. ANL treatment increased hypodiploidy and decreased the number of HepG2 cell in G0 -G1 phase at both 24 and 48 hours. Furthermore, ANL increased the level of apoptosis in both HepG2 and HT-29 cells in a time-dependent manner via enhanced production of reactive oxygen species and altered mitochondrial membrane potential, indicative of intrinsic apoptotis pathway activation. Immunoblot analysis confirmed the time-dependent elevation of levels of cytochrome c, initiator caspase-9 and activation of caspase-3. ANL immunohistochemistry on colon cancer tissue and quantification of AFP in HCC patient serum samples by developing an ANL-anti-AFP antibody sandwich enzyme-linked immunosorbent assay confirmed the diagnostic potential of ANL. Here, interaction of ANL with AFP could be effectively blocked in the presence of competing fucose-bearing glycans. We found ANL to be more sensitive than Lens culinaris lectin, a well-known fucose-specific lectin and currently used diagnostic agent. ANL can be further explored as a diagnostic and anti-cancer agent.

Galactosamine (GalN) is a well-known agent for inducing viral hepatitis models in rodents, but it can cause toxicity on different organs. Vitamin U (Vit U) has been proved as a powerful antioxidant on many toxicity models. The present study was designed to investigate the protective effects of Vit U on GalN-induced stomach injury. Rats were divided into four groups as follows: control (group I), Vit U given animals (50 mg/kg per day; group II), GalN administered animals (500 mg/kg at a single dose; group III), GalN + Vit U given animals (at the same dose and time, group IV). At the end of the 3rd day, animals were killed, and stomach tissues were taken. They were homogenized and centrifuged. In comparison to the control group, glutathione, total antioxidant capacity levels, catalase, superoxide dismutase, glutathione peroxidase, glutathione reductase, glutathione-S-transferase, and Na

A fucoidan, sulfated polysaccharide, was extracted from the brown seaweed Cystoseira schiffneri during 4 harvest periods (December, April, July, and September) and studied for its structural and chemical properties. The Cystoseira schiffneri fucoidan (CSF) showed important variation in sulfate content ranging from 7.8% in December to 34.8% in July. This was confirmed by Fourier transform infrared and nuclear magnetic resonance spectroscopies showing characteristic signals of sulfated polysaccharides. Molecular mass of the CSF varied as a function of season from 3,745 in December to 26,390 Da in July. Gas chromatography–mass spectroscopy showed that CSF fractions were “mannogalactofucans” composed mainly of mannose, fucose, and galactose with low levels of other monosaccharides. Moreover, interesting in vitro antioxidant activities that depend on the harvest season were noted for CSF. Thus, the present work might contribute to establish criteria for extracting bioactive fucoidans from an endemic Tunisian seaweed C. schiffneri., A novel fucoidan, mannogalactofucan, was extracted from the brown seaweed Cystoseira schiffneri. Mw of extracted fucoidans increased from 3,745 at December to 26,390 Da at July. The fucoidans showed interesting in vitro antioxidant activities that depend on the harvest season.

Drug resistance represents an obstacle in colorectal cancer (CRC) treatment because of its association with poor prognosis. rBC2LCN is a lectin isolated from Burkholderia that binds cell surface glycans that have fucose moieties. Because fucosylation is enhanced in many types of cancers, this lectin could be an efficient drug carrier if CRC cells specifically present such glycans. Therefore, we examined the therapeutic efficacy and toxicity of lectin drug conjugate therapy in CRC mouse xenograft models. The affinity of rBC2LCN for human CRC cell lines HT‐29, LoVo, LS174T, and DLD‐1 was assessed in vitro. The cytocidal efficacy of a lectin drug conjugate, rBC2LCN‐38 kDa domain of pseudomonas exotoxin A (PE38) was evaluated by MTT assay. The therapeutic effects and toxicity for each CRC cell line‐derived mouse xenograft model were compared between the intervention and control groups. LS174T and DLD‐1 cell lines showed a strong affinity for rBC2LCN. In the xenograft model, the tumor volume in the rBC2LCN‐PE38 group was significantly reduced compared with that using control treatment alone. However, the HT‐29 cell line showed weak affinity and poor therapeutic efficacy. No significant toxicities or adverse responses were observed. In conclusion, we demonstrated that rBC2LCN lectin binds CRC cells and that rBC2LCN‐PE38 significantly suppresses tumor growth in vivo. In addition, the efficacy of the drug conjugate correlated with its binding affinity for each CRC cell line. These results suggest that lectin drug conjugate therapy has potential as a novel targeted therapy for CRC cell surface glycans., rBC2LCN lectin showed affinity for colorectal cancer (CRC) cell surface. rBC2LCN‐PE38 suppressed subcutaneous CRC tumor growth rates in mouse models. Lectin drug conjugate therapy could be a novel therapeutic option for CRC.

A general and efficient strategy for synthesis of tri‐, hexa‐ and heptasaccharidic substructures of the lipopolysaccharide of Providencia rustigianii O34 is described. For the heptasaccharide seven different building blocks were employed. Special features of the structures are an α‐linked galactosamine and the two embedded α‐fucose units, which are either branched at positions‐3 and ‐4 or further linked at their 2‐position. Convergent strategies focused on [4+3], [3+4], and [4+2+1] couplings. Whereas the [4+3] and [3+4] coupling strategies failed the [4+2+1] strategy was successful. As monosaccharidic building blocks trichloroacetimidates and phosphates were employed. Global deprotection of the fully protected structures was achieved by Birch reaction., Embedded fucoses: First syntheses of substructures of the O‐polysaccharide of Providencia rustiginii O34 are presented. The repeating unit is a heptasaccharide with seven different monosacchride units (see figure).

Enterovirus 71 (EV71) is the predominant pathogen for severe hand, foot, and mouth disease (HFMD) in children younger than 5 years, and currently no effective drugs are available for EV71. Thus, there is an urgent need to develop new drugs for the control of EV71 infection. In this study, LJ04 was extracted from Laminaria japonica using diethylaminoethyl cellulose-52 with 0.4 mol/l NaCl as the eluent, and its virucidal activity was evaluated based on its cytopathic effects on a microplate. LJ04 is composed of fucose, galactose, and mannose and mainly showed good virucidal activity against EV71. The antiviral mechanisms of LJ04 were the direct inactivation of the virus, the blockage of virus binding, disruptions to viral entry, and weak inhibitory activity against the nonstructural protein 3C. The two most important findings from this study were that LJ04 inhibited EV71 proliferation in HM1900 cells, which are a human microglia cell line, and that LJ04 can directly inactivate EV71 within 2 hr at 37°C. This study demonstrates for the first time the ability of a polysaccharide from L. japonica to inhibit viral and 3C activity; importantly, the inhibition of 3C might have a minor effect on the antiviral effect of LJ04. Consequently, our results identify LJ04 as a potential drug candidate for the control of severe EV71 infection in clinical settings.

Water‐soluble polysaccharides (LCPs) were extracted from litchi, wherein LPS, LPH, LPP and LPM were extracted by ultrasound, hot water, ultra‐high pressure and microwave methods, respectively. The physicochemical properties, components, structures and in vitro antioxidant activities of four LCPs were investigated. LPP was the highest in yield and purity, and most physicochemical properties of four LCPs were similar. FTIR showed that LPP, LPS and LPH had ɑ‐dominating configuration in pyranose rings. SEM showed that four LCPs were different, and LPH and LPP had relatively smooth and compact surfaces. The Mw of LPS, LPH, LPP and LPM were 79 538, 320 213, 192 891 and 200 198 Da, respectively. All four LCPs consisted of fucose, galactose, glucose and rhamnose. Except for LPS, the other three LCPs contained mannose. Based on the results of in vitro antioxidant activity, 5 mg mL⁻¹ LPP presented the highest in DPPH and hydroxyl scavenging activity, while it ranked secondly in reducing power.

2'-Fucosyllactose (2-FL), one of the most abundant oligosaccharides in human milk, has been spotlighted for its neutraceutical and pharmaceutical potentials. Microbial production of 2-FL is promising since it is efficient as compared to other production methods. In 2-FL microbial production via the salvage pathway for biosynthesis of guanosine 5'-diphosphate (GDP)-l-fucose from fucose, the conversion yield from fucose is important because of the high price of fucose. In this study, deletion of the genes (araA and rhaA) coding for arabinose isomerase (AraA) and rhamnose isomerase (RhaA) was attempted in engineered Escherichia coli for improving 2-FL production by using fucose, lactose, and glycerol. The engineered E. coli constructed previously is able to express fucokinase/GDP-l-fucose pyrophosphorylase (Fkp) from Bacteroides fragilis and the α-1,2-fucosyltransferase (FucT2) from Helicobacter pylori and deficient in β-galactosidase (LacZ), fucose isomerase (FucI), and fuculose kinase (FucK). The additional double-deletion of the araA and rhaA genes in the engineered E. coli enhanced the product yield of 2-FL to 0.52 mole 2-FL/mole fucose, and hence the concentration of 2-FL reached to 47.0 g/L, which are 44% and two-fold higher than those (23.1 g/L and 0.36 mole 2-FL/mole fucose) of the control strain in fed-batch fermentation. Elimination of sugar isomerases exhibiting promiscuous activities with fucose might be critical in the microbial production of 2-FL through the salvage pathway of GDP-l-fucose.

Despite the advantages of transplantation of umbilical cord blood's (UCB's) hematopoietic stem cells (uHSCs) for hematologic malignancy treatment, there are two major challenges in using them: (a) Insufficient amount of uHSCs in a UCB unit; (b) a defect in uHSCs homing to bone marrow (BM) due to loose binding of their surface glycan ligands to BM's endothelium selectin receptors. To overcome these limitations, after poly l-lactic acid (PLLA) scaffold establishment and incubation of uHSCs with fucosyltransferase-VI and GDP-fucose, ex vivo expansion of these cells on selectin-coated scaffold was done. The characteristics of the cultured fucosylated and nonfucosylated cells on a two-dimensional culture system, PLLA, and a selectin-coated scaffold were evaluated by flow cytometry, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, colony-forming unit (CFU) assay, and CXCR4 expression at the messenger RNA and protein levels. According to the findings of this study, optimized attachment to the scaffold in scanning electron microscopy micrograph, maximum count of CFU, and the highest 570 nm absorption were observed in fucosylated cells expanded on selectin-coated scaffolds. Furthermore, real-time polymerase chain reaction showed the highest expression of the CXCR4 gene, and immunocytochemistry data confirmed that the CXCR4 protein was functional in this group compared with the other groups. Considered together, the results showed that selectin-coated scaffold could be a supportive structure for fucosylated uHSC expansion and homing by nanotopography. Fucosylated cells placed on the selectin-coated scaffold serve as a basal surface for cell-cell interaction and more homing potential of uHSCs. Accordingly, this procedure can also be considered as a promising technique for the hematological disorder treatment and tissue engineering applications.

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

Rationale Glycoprotein fucosylation, one of the major posttranslational modifications, is known to be highly involved in proteins related to various cancers. Fucosylation occurs in the core and/or outer sites of N-glycopeptides. Elucidation of the fucosylation type of N-glycoproteins is therefore important. However, it has remained a challenge to classify the fucosylation types of N-glycopeptides using collision-induced dissociation (CID) tandem mass (MS/MS) spectra. Methods The relative intensities of the Y1 F, Y2 F, Y3 F, and Y4 F product ions in the CID-MS/MS spectra of the IgG N-glycopeptides were measured for core fucosylation. The Core Fucose Index (CFI) was then calculated by multiplication of the relative intensities with a weight factor from logistic regression to differentiate between the core and none fucosylation. From the relative intensities of the B2 F and B3 SF ions of the MS/MS spectra of the AGP N-glycopeptides for outer fucosylation, the Outer Fucose Index (OFI) was calculated to differentiate between the outer and none fucosylation. Results In order to classify core and/or outer fucosylation of N-glycoproteins, we defined the fucosylation score (F-score) by a sigmoidal equation using a combination of the CFI and the OFI. For application, we classified the fucosylation types of N-glycoproteins in human plasma with 99.7% accuracy from the F-score. Human plasma samples showed 54.4%, 33.3%, 10.3%, and 1.6% for none, core, outer, and dual fucosylated N-glycopeptides, respectively. Core fucosylation was abundant at mono- and bi-antennary N-glycopeptides. Outer fucosylation was abundant at tri- and tetra-antennary N-glycopeptides. In total, 113 N-glycopeptides of 29 glycoproteins from 3365 glycopeptide spectral matches (GPSMs) were classified for different types of fucosylation. Conclusions We established an F-score to classify three different fucosylation types: core, outer, and dual types of N-glycopeptides. The fucosylation types of 20 new N-glycopeptides from 11 glycoproteins in human plasma were classified using the F-score. Therefore, the F-score can be useful for the automatic classification of different types of fucosylation in N-glycoproteins of biological fluids including plasma, serum, and urine.

Progress has been made in overcoming antibody-mediated rejection of porcine xenografts by deleting pig genes that produce unique carbohydrate epitopes. Pigs deficient in galactose α-1,3 galactose (gene modified: GGTA1) and neu5Gc (gene modified: CMAH) have reduced levels of human antibody binding. Previously we identified α-fucose as a glycan that was expressed in high levels on cells of GGTA1/CMAH KO pigs. To validate the α-fucose phenotype observed previously we compared lectin affinity toward human and pig serum glycoproteins by dot blot analysis and confocal microscopy. Human anti-fucose antibody isolated by affinity chromatography was tested for specificity to L-fucose by custom macroarray. The affinity and cytotoxicity of the isolated human anti-fucose antibody toward human and GGTA1/CMAH KO pig PBMCs was determined by flow cytometry. Dot blot and confocal analysis support out previous findings that α-fucose is more highly expressed in pigs than humans. Pig kidney glomeruli and tubules contain abundant α-fucose and may represent focal sites for anti-α-fucose antibody binding. The Isolated human anti-fucose IgA, IgG and IgM bound to GGTA1/CMAH KO pig PBMC and were cytotoxic. Interestingly, the isolated human IgG cross reacted with the methyl pentose, L-rhamnose. Human anti-fucose antibody bound and was cytotoxic to GGTA1/CMAH KO pig peripheral blood monocytes. We have shown that α-fucose is an abundant target for cytotoxic human antibody in the organs of genetically modified pigs important to xenotransplantation.

Difucosyllactose (Di-FL) has strong antimicrobial activity against various pathogens, including group B Streptococcus, identified as the leading cause of neonatal sepsis. In this study, we sought to develop Escherichia coli as a microbial cell factory for efficiently producing Di-FL as well as 2'-fucosyllactose (2'-FL), the most abundant fucosylated oligosaccharide in human milk, by utilizing the salvage guanosine 5'-diphosphate (GDP)-l-fucose biosynthetic pathway.The biosynthetic pathway for producing fucosylated oligosaccharides via the salvage pathway requires two enzymes, l-fucokinase/GDP-l-fucose phosphorylase (FKP) from Bacteroides fragilis and α-1,2-fucosyltransferase (FucT2) from Helicobacter pylori. To decrease the intracellular accumulation of 2'-FL while increasing substrate accessibility to FKP and FucT2, we evaluated whether extracellular secretion of FKP and FucT2 would enhance the production of fucosylated oligosaccharides. Among various engineered strains constructed in this study, the ΔLFAR-YA/FF+P-PLAThis study highlights the benefits of extracellular secretion of enzymes to improve biotransformation efficiency, as the transport of substrates and/or products across the cell membrane is limited.

FleA (or AFL), a fucose lectin, was recently identified in the opportunistic mold Aspergillus fumigatus, which causes fatal lung infections in immunocompromised patients. We designed di-, hexa- and octavalent fucosides with various spacer arm lengths to block the hexameric FleA through chelation. Microcalorimetry measurements showed that the ethylene glycol (EG) spacer arm length has a strong influence on the binding affinity of the divalent fucosides. The relationship between the EG length and chelate binding efficiency to FleA was explored according to polymer theory. Hexa- and octavalent compounds based on cyclodextrin and octameric silsesquioxane scaffolds were nanomolar FleA inhibitors, surpassing their monovalent fucose analogue by more than three orders of magnitude. Importantly, some of the fucosides were highly efficient in preventing fungal spore adhesion to bronchoepithelial cells, with half maximal inhibitory concentration values in the micromolar range. We propose that the synergistic antiadhesive effect observed can be ascribed to chelate binding to FleA and to the formation of conidium aggregates, as observed by optical microscopy. These fucosides are promising tools that can be used to better understand the role of FleA in conidia pathogenicity and host defenses against invasive aspergillosis.

Plant biotechnology journal 17(2), 350-361 (2019). doi:10.1111/pbi.12981, Published by Wiley-Blackwell, Oxford

Live cell imaging of protein-specific glycoforms holds great promise for revolutionizing the study of glycochemistry. The imaging protocols developed thus far build upon the paired interplay of probe units, thus limiting the number of monosaccharide identification channels. A hierarchical coding (HieCo) imaging strategy, with DNA coding and decoding of protein and monosaccharides executed in fidelity to the hierarchical order of target glycoprotein, is reported herein and features expandable monosaccharide identification channels. A proof-of-concept protocol has been developed for MUC1-specific imaging of terminal sialic acid (Sia) and fucose (Fuc) on MCF-7, T47D, MDA-MB-231, and PANC-1 cells, revealing distinct monosaccharide patterns for four types of cells. The protocol also permits dynamic monitoring of changes in MUC1-specific monosaccharide patterns associated with both the alteration of cellular physiological states and the occurrence of a biologically important process.

Carbohydrates and their conjugates play important roles in many biological processes including fertilization, differentiation, development, immune response, and infection. Their activities are largely dependent on the properties of terminal mono- or disaccharides. Galactose, mannose, fucose, glucose, sialic acid, etc., are commonly used as powerful scaffolds installed on drug molecules for targeting specific tissues including brain, liver, and cancers, and as epitopes for enhancing the targeting of various vaccines. This review focuses on the influence of their structural variations, including changes in sugar type, substituent groups and their positions, as well as length of linker portion, on the targeting of drugs or their efficacy. Particular attention is paid to the targeting properties of mono- and disaccharides applied in drug design and discovery.

Recombinant butyrylcholinesterase produced in a metabolically regulated transgenic rice cell culture (rrBChE) was purified to produce a highly pure (95%), active form of enzyme. The developed downstream process uses common manufacturing friendly operations including tangential flow filtration, anion-exchange chromatography, and affinity chromatography to obtain a process recovery of 42% active rrBChE. The purified rrBChE was then characterized to confirm its comparability to the native human form of the molecule (hBChE). The recombinant and native enzyme demonstrated comparable enzymatic behavior and had an identical amino acid sequence. However, rrBChE differs in that it contains plant-type complex N-glycans, including an α-1,3 linked core fucose, and a β-1,2 xylose, and lacking a terminal sialic acid. Despite this difference, rrBChE is demonstrated to be an effective stoichiometric bioscavenger for five different organophosphorous nerve agents in vitro. Together, the efficient downstream processing scheme and functionality of rrBChE confirm its promise as a cost-effective alternative to hBChE for prophylactic and therapeutic use.

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

The nucleotide sugar guanosine 5’-diphospho-β-L-galactose (GDP-L-Gal) is known as a key intermediate of L-ascorbic acid biosynthesis pathway of plant and algae. In addition, GDP-L-Gal serves as a donor substrate of L-galactosyltransferase, which transfers L-Gal on the non-reducing ends of glycoconjugates. To synthesize varieties of L-Gal containing glycoconjugates and explore the novel L-galactosyltransferases, GDP-L-Gal needs to be prepared since it is not commercially available. In plants, GDP-D-mannose-3’,5’-epimerase (GME) converts GDP-α-D-mannose (GDP-D-Man) to GDP-L-Gal and GDP-L-gulose. GDP-L-Gal has been previously prepared using GME and GDP-D-Man, which equilibrium ratio was biased to GDP-D-Man. In this study, the efficient GDP-L-Gal production from L-Gal was established using bacterial L-fucokinase/GDP-L-fucose pyrophosphorylase (FKP), L-Fuc, adenosine 5’-triphosphate (ATP) and guanosine 5’-triphosphate (GTP). High synthesis yield was obtained after screening of the optimum FKP reaction conditions in a 96-well format and analysis by multiplexed capillary electrophoresis (MP-CE). Conversion of L-Gal substrate yielded 97% GDP-L-Gal using purified recombinant FKP expressed in E. coli Moreover, GDP-L-Gal was successfully purified with 92% (34.5 mg) overall yield from the FKP reaction mixture. GDP-L-Gal is now readily available for studies on L-galactosyltransferases and L-fucosyltransferases.

Biofilms of sulfate-reducing bacteria (SRB) are often the major cause of microbiologically influenced corrosion. The representative SRB Desulfovibrio vulgaris has previously been shown to have a biofilm that consists primarily of protein. In this study, by utilizing lectin staining, we identified that the biofilm of D. vulgaris also consists of the matrix components mannose, fucose and N-acetylgalactosamine (GalNAc), with mannose predominating. Based on these results, we found that the addition of mannose and the nonmetabolizable mannose analog 2-deoxy-d-glucose inhibits the biofilm formation of D. vulgaris as well as that of D. desulfuricans; both compounds also dispersed the SRB biofilms. In addition, the enzyme N-acetylgalactosaminidase, which degrades GalNAc, was effective in dispersing D. vulgaris biofilms. Therefore, by determining composition of the SRB biofilm, effective biofilm control methods may be devised.

The use of bone marrow-derived mesenchymal stem cells (MSCs) for clinical and experimental studies is increasing, but full characterization of MSCs in veterinary species is hindered by the variability in species-specific cell surface marker expression and antibody cross reactivity. Recent studies demonstrated that the glycans in the glycocalyx of MSCs are promising candidates as cell biomarkers. In the present study, we analyzed the glycocalyx of canine MSCs (cMSCs), ovine MSCs (oMSCs), and equine MSCs (eMSCs) using a cell microarray procedure in which MSCs were spotted on microarray slides and incubated with a panel of 14 biotinylated lectins and Cy3-conjugated streptavidin. The signal intensity was then detected using a microarray scanner. The lectin-binding signals indicated that the MSC surface of the investigated species contained both N- and O-linked glycan types, with N-glycosylation predominating over O-glycosylation and fucosylation being more abundant than sialylation. Relative quantification revealed an interspecific difference between these glycans. In addition, cMSCs expressed more α2,3-linked sialic acid (MAL II), terminal lactosamine (RCA120 ), and α1,6 and α1,3 fucosylated oligosaccharides (PSA, LTA); oMSCs exhibited more T antigen (Jacalin), GalNAcα1,3(LFucα1,2)Galβ1,3/4GlcNAcβ1 (DBA), chitotriose (succinylated WGA), and α1,2-linked fucose (UEA I); and eMSCs showed a higher density of α2,6 sialic acids (SNA) and high mannose N-glycans (Con A). Using cell microarray methodology, we have for the first time demonstrated differences in the glycosylation profiles of cMSC, oMSC, and eMSC surfaces. These results could be valuable as resources and references for MSC differentiation and molecular remodeling in clinical cell-based therapy and tissue engineering studies. © 2017 International Society for Advancement of Cytometry.

Summary Litchi (Litchi chinensis Sonn.) is a fruit containing many nutrients. Based on a three level, three-variable Box-Behnken design, response surface methodology, was used to optimise the process of ultra-high pressure (UHP) assisted extraction of litchi polysaccharides (LCP). The optimal extraction conditions were as follows: pressure 460 MPa, treatment time 17 min and liquid–solid ratio 15. The experimental yield for extracting LCP was 12.01% under the optimal conditions. The LCP had a high polysaccharide and low protein content. GC results showed that LCP was composed of rhamnose, fucose, mannose and galactose with the corresponding molar percentages of 7.89, 46.45, 9.71 and 19.63. Infrared analysis showed that LCP had a typical spectrum of an α-configuration pyranose ring. Antioxidant activity analysis revealed that LCP possessed a strong reducing power and 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity and could protect against the damage of hydroxyl radicals. The investigations demonstrated that UHP could be a valuable way to separate polysaccharides from litchi pulp, and LCP may be potentially developed to an antioxidant for food industries.

To engineer a host cell line that produces defucosylated mAbs with superior antibody-dependent cellular cytotoxicity, we disrupted α-1, 6 fucosyltransferase (FUT8) gene in CHO-S (CHO is Chinese hamster ovary) cells by clustered regularly interspaced short palindromic repeats-CRISPR associated nuclease 9. The gene knockout cell line was evaluated for growth, stability, and product quality. The growth profile of FUT8 gene knockout CHO-S (FUT8 -/-) cells was comparable with wild type CHO-S cells. FUT8 catalyzes the transfer of a fucose residue from GDP-fucose to N-glycans residue. Defucosylated IgG1 antibodies produced by FUT8 -/- cells showed increased binding affinities to human FcγRIIIa and higher activities in mediating antibody-dependent cellular cytotoxicity, comparing with conventional fucosylated IgG1. Our results demonstrated the potential of using the clustered regularly interspaced short palindromic repeats-CRISPR associated nuclease 9 technology in cell line engineering for biopharmaceutical industrial applications.

Streptococcus pneumoniae harbors a significant number of transporters, including phosphotransferase (PTS) systems, allowing the bacterium to utilize a number of different carbohydrates for metabolic and other purposes. The genes encoding for one PTS transport system in particular (EIIfuc ) are found within a fucose utilization operon in S. pneumoniae TIGR4. Here, we report the three-dimensional structures of IIAfuc and IIBfuc providing evidence that this PTS system belongs to the EIIman family. Additionally, the predicted metabolic pathway for this distinctive fucose utilization system suggests that EIIfuc transports the H-disaccharide blood group antigen, which would represent a novel PTS transporter specificity. Proteins 2017; 85:963-968. © 2016 Wiley Periodicals, Inc.

Summary Plant‐produced glycoproteins contain N‐linked glycans with plant‐specific residues of β(1,2)‐xylose and core α(1,3)‐fucose, which do not exist in mammalian‐derived proteins. Although our experience with two enzymes that are used for enzyme replacement therapy does not indicate that the plant sugar residues have deleterious effects, we made a conscious decision to eliminate these moieties from plant‐expressed proteins. We knocked out the β(1,2)‐xylosyltranferase (XylT) and the α(1,3)‐fucosyltransferase (FucT) genes, using CRISPR/Cas9 genome editing, in Nicotiana tabacum L. cv Bright Yellow 2 (BY2) cell suspension. In total, we knocked out 14 loci. The knocked‐out lines were stable, viable and exhibited a typical BY2 growing rate. Glycan analysis of the endogenous proteins of these lines exhibited N‐linked glycans lacking β(1,2)‐xylose and/or α(1,3)‐fucose. The knocked‐out lines were further transformed successfully with recombinant DNaseI. The expression level and the activity of the recombinant protein were similar to that of the protein produced in the wild‐type BY2 cells. The recombinant DNaseI was shown to be totally free from any xylose and/or fucose residues. The glyco‐engineered BY2 lines provide a valuable platform for producing potent biopharmaceutical products. Furthermore, these results demonstrate the power of the CRISPR/Cas9 technology for multiplex gene editing in BY2 cells.

Scope 2´-Fucosyllactose (2´FL) is an abundant oligosaccharide in human milk. It is hypothesized that its brain enrichment is associated with improved learning. Accumulation of 2´FL in organs, biological fluids, and feces is assessed in wild-type and germ-free mice. Methods and results 13 C-labelled 2´FL is applied to NMRI wild-type mice intravenously (0.2 g kg-1 ) or orally (1 g kg-1 ), while controls receive saline. Biological samples are collected (0.5-15 h) and 13 C-enrichment is measured by elemental analysis isotope ratio mass spectrometry (EA-IRMS). After oral application, 2´FL is primarily eliminated in the feces. 13 C-enrichment in organs including the brain follows the same pattern as in plasma with a maximum peak after 5 h. However, 13 C-enrichment is only detected when the 13 C-2´FL bolus reaches the colon. In contrast, in germ-free mice, the 13 C-bolus remains in the intestinal content and is expelled via the feces. Furthermore, intravenously applied 13 C-2´FL is eliminated via urine; no 13 C-enrichment of organs is observed, suggesting that intact 2´FL is not retained. Conclusions 13 C-enrichment in brain and other organs after oral application of 13 C-2´FL in wild-type mice indicates cleaved fucose or other gut microbial 2´FL metabolites may be incorporated, as opposed to intact 2´FL.

The extent of afucosylation, which refers to the absence of core fucose on Fc glycans, can correlate positively with the antibody-dependent cellular cytotoxicity (ADCC) activity of a monoclonal antibody (mAb). Therefore, it is important to maintain consistent afucosylation during cell culture process scale-up in bioreactors for a mAb with ADCC activity. However, there is currently a lack of understanding about the impact of partial pressure of carbon dioxide (pCO2 )-a parameter that can vary with bioreactor scale-on afucosylation. Using a small-scale (3 L) bioreactor model that can modulate pCO 2 levels through modified configurations and gassing strategies, we identified three cell culture process parameters that influence afucosylation of a mAb produced by a recombinant Chinese Hamster Ovary (CHO) cell line: pCO 2 , media hold duration (at 37°C), and manganese. These three-independent parameters demonstrated a synergistic effect on mAb afucosylation; increase in pCO 2 , media hold duration, and manganese consistently increased afucosylation. Our investigations into the underlying mechanisms through proteomic analysis indicated that the synergistic interactions downregulated pathways related to guanosine diphosphate-fucose synthesis and fucosylation, and upregulated manganese transport into the CHO cells. These new findings highlight the importance of considering potential differences in culture environment and operations across bioreactor scales, and understanding the impact of their interactions on product quality.

L-Fucose (6-deoxy-L-galactose) is a major constituent of glycans and glycolipids in mammals. Fucosylation of glycans can confer unique functional properties and may be an economical way to manufacture L-fucose. Research can extract L-fucose directly from brown algae, or by enzymatic hydrolysis of L-fucose-rich microbial exopolysaccharides. However, these L-fucose production methods are not economical or scalable for various applications. We engineered an Escherichia coli strain to produce L-fucose. Specifically, we modified the strain genome to eliminate endogenous L-fucose and lactose metabolism, produce 2'-fucosyllactose (2'-FL), and to liberate L-fucose from 2'-FL. This E. coli strain produced 16.7 g/L of L-fucose with productivity of 0.1 g·L-1 ·h-1 in a fed-batch fermentation. This study presents an efficient one-pot biosynthesis strategy to produce a monomeric form of L-fucose by microbial fermentation, making large-scale industrial production of L-fucose feasible.

PURPOSE Fucosylation of alpha-fetoprotein (AFP) is closely correlated with the diagnosis of patients with hepatocellular carcinoma (HCC). In current, a micro-total analysis system (μTAS) using immunoassay has been developed for determining fucosylated AFP EXPERIMENTAL DESIGN: We compared two analytical methods, μTAS and liquid chromatography-parallel reaction monitoring mass spectrometry (LC-PRM MS), for the measurement of fucosylated AFP in serum to evaluate the usefulness of the results. For this purpose, serum samples were used (cirrhosis, n = 105; HCC, n = 105), and we have discussed the analytical performance of these two methods RESULTS: We observed a correlation (R2 = 0.84) between LC-PRM MS and μTAS using samples where fucosylated levels were measured by both methods. The fucosylated level of AFP by LC-PRM MS better differentiated between cirrhosis and HCC patients than those by μTAS (AUC = 0.910 vs. 0.861), particularly in subgroups with a level of total AFP

Catalysts that promote carbohydrate degradation have a wide range of potential applications, but the use of either enzyme glycosidases or small-molecule catalysts in biological systems raises significant challenges. Herein, we demonstrate a novel strategy for the design of synthetic agents that mimic natural glycosidases and address current problems for biological use. This strategy is illustrated by application to the development of potential blood substitutes for the rare Bombay blood type that is characterized by a deficiency of H2 antigen. Metallopeptides with 16 to 20 amino acids were constructed as artificial fucosidases that exhibit selective carbohydrate cleavage reactivity toward l-fucose over d-glucose. Selective fucose cleavage from the H2-antigen saccharide enables efficient removal of H2 antigen from erythrocytes and thereby accomplishes the conversion of regular human type-O blood into a potential blood substitute for the rare Bombay blood type.

Seven derivatives of L-fucose: methyl α-L-fucopyranoside, methyl 2-O-methyl-α-L-fucopyranoside, methyl 3-O-methyl-α-L-fucopyranoside, methyl 4-O-methyl-α-L-fucopyranoside, α-L-fucopyranosyl-(1→4)-α/β-L-rhamnopyranose, methyl α-L-fucopyranosyl-(1→4)-α-L-rhamnopyranoside and methyl α-L-fucopyranosyl-(1→4)-β-L-rhamnopyranoside, were chemically synthesized and characterized. The derivatives of L-fucose were designed to mimic the L-fucose moieties present in the A-chain of pectic rhamnogalacturonan-II and used to investigate the regiospecificity of three heterologously expressed xylosyltransferases from Arabidopsis thaliana. The monosaccharide methyl α-L-fucopyranoside was the most efficient acceptor, 30 fold more effective than methyl 4-O-methyl-α-L-fucopyranoside. O-methylation in position 2 or 3 abolished acceptor function. The efficiencies of the disaccharides as acceptors were methyl α-L-fucopyranosyl-(1→4)-β-L-rhamnopyranoside > α-L-fucopyranosyl-(1→4)-α/β-L-rhamnopyranose > methyl α-L-fucopyranosyl-(1→4)-α-L-rhamnopyranoside. These findings demonstrate the utility of acceptor derivatives as probes of glycosyltransferase regiospecificity.

Methods involving the use of mathematical models of competitive ligand-receptor binding to characterize mixtures of ligands in terms of compositions and properties of the component ligands have been developed. The associated mathematical equations explicitly relate component ligand physical-chemical properties and mole fractions to measurable properties of the mixture including steady state binding activity, 1/Kd,apparent or equivalently 1/EC50, and kinetic rate constants kon,apparent and koff,apparent allowing: (1) component ligand physical property determination and (2) mixture property predictions. Additionally, mathematical equations accounting for combinatorial considerations associated with ligand assembly are used to compute ligand mole fractions. The utility of the methods developed is demonstrated using published experimental ligand-receptor binding data obtained from mixtures of afucosylated antibodies that bind FcγRIIIa (CD16a) to: (1) extract component ligand physical property information that has hitherto evaded researchers, (2) predict experimental observations, and (3) provide explanations for unresolved experimental observations. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:500-510, 2017.

Novel polymeric cell adhesion inhibitors were developed in which the selectin tetrasaccharide sialyl-LewisX (SLeX ) is multivalently presented on a biocompatible poly(2-hydroxypropyl)methacrylamide (PHPMA) backbone either alone (P1) or in combination with O-sulfated tyramine side chains (P2). For comparison, corresponding polymeric glycomimetics were prepared in which the crucial "single carbohydrate" substructures fucose, galactose, and sialic acid side chains were randomly linked to the PHPMA backbone (P3 or P4 (O-sulfated tyramine)). All polymers have an identical degree of polymerization, as they are derived from the same precursor polymer. Binding assays to selectins, to activated endothelial cells, and to macrophages show that polyHPMA with SLeX is an excellent binder to E-, L-, and P-selectins. However, mimetic P4 can also achieve close to comparable binding affinities in in vitro measurements and surprisingly, it also significantly inhibits the migration of macrophages; this provides new perspectives for the therapy of severe inflammatory diseases.