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High-affinity monovalent ligands for lectins are challenging to develop due to weak binding interactions. This study investigates the potential of rationally designed covalent ligands targeting the N-terminal domain of BC2L-C lectin from Burkholderia cenocepacia , a pathogen causing severe respiratory infections in immunocompromised patients. Antiadhesion therapy is emerging as a complementary approach against such infections, and bacterial lectins are suitable targets. The fucose-specific BC2L-C-Nt recognizes blood group oligosaccharides on host cells. Using a computational approach, we designed reversible covalent competitive ligands that include a fucoside anchor and a salicylaldehyde warhead targeting Lys108 near the fucose-binding site. Several candidates were synthesized and tested using competition experiments. The most effective ligand improved the IC 50 of methyl-fucoside by 2 orders of magnitude, matching the affinity of the native H-type 1 trisaccharide. Control experiments confirmed the importance of both fucose anchor and salicylaldehyde moiety in the ligand's affinity. Mass analysis confirmed the covalent interaction with Lys108.

Deoxy-hexose sugars, such as rhamnose and quinovose, and the dideoxy-hexoses colitose, abequose, and tyvelose are highly antigenic given that they are absent from animal glycoconjugates. To investigate the specificity of antibodies towards structurally similar carbohydrate epitopes found in bacteria, we synthesized trisaccharides containing colitose, abequose, and fucose motifs. Each trisaccharide was designed with a spacer ending with a primary amino group. These trisaccharide constructs were immobilized on O-succinimide coated glass slides alongside bacterial lipopolysaccharides (LPS) containing colitose, abequose, and fucose residues. We compared the recognition of the synthetic trisaccharides and natural LPS including structurally related epitopes by monoclonal and polyclonal antibodies targeting bacterial LPS. Additionally, we used arrays displaying the synthetic trisaccharides and natural LPS to assess the variability of IgA reactivity from breast milk samples towards the carbohydrate antigens. The results obtained underlined the cross-reactivity of polyclonal antibodies towards structurally related carbohydrate antigens and revealed a broad reactivity of breast milk-derived IgA towards the carbohydrate antigens tested. The significant cross-reactivity of antibodies towards structurally related LPS antigens may lead to false-positive detection of bacterial serotypes when used for diagnostic purposes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Biological glycans mediate several physiological processes, thus altered glycosylation patterns can lead to different diseases such as autoimmune, infectious, chronic anti-inflammatory diseases, or even cancer. In fact, alterations in fucosylation in either N- or O-glycans are among the most frequent changes in glycosylation patterns associated with cancer. Therefore, elucidation of the role of glycoconjugate glycans is essential for understanding the development of pathologies where they are involved. In this sense glycosidases are excellent tools, since they catalyse the selective removal of sugar residues, allowing the evaluation of changes in their biological role due to glycan removal. This work describes the purification and characterization of a α-fucosidase from the fungus Dichostereum sordulentum 1488. It is a homodimer with a molecular weight of 214 kDa and optimum pH and temperature of 4.0 and 70 °C respectively. It has a K M of 0.27 mM and V Max of 3.3 μmoles PNP/min per mg for the substrate p-nitrophenyl-α-l-fucopyranoside, showing a substrate inhibition profile. It showed high specificity for the hydrolysis of fucose linked by α-(1,2) bonds. The identification, purification, and characterization of this new α-fucosidase is highly relevant for enlarging the availability of glycosidases for use as tools for glycan elucidation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: The stem of Abroma augusta contains mucilaginous polysaccharides having numerous ethnomedicinal properties. The present work aimed to develop a scalable ultrasonic-assisted aqueous Abroma augusta mucilage (AAM) extraction (UAE) method and further explores its emulsifying property and toxicity concern., Results: The combination of ultrasonic power (750 W), solid-to-liquid ratio (1:15) and temperature (348 K) gave the highest extraction yield of 2.28% with a diffusivity value of 3.85 × 10 -9  m 2  s -1 , which was higher than aqueous extraction method using a kinetic model based on Fick's second law of diffusion. The extracted polysaccharide showed no toxicity as measured through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assay on RAW cell line. Additionally, the polysaccharide over its critical micelle concentration (400, 500, 600 and 700 μg mL -1 ) offered emulsifying properties with 0.5%, 1% and 5% oil (v/v). The emulsion with a polysaccharide concentration of 600 μg mL -1 with 5% oil (v/v) provides stability against coalescence for 3 days., Conclusion: The overall findings indicated that UAE of AAM polysaccharide can be used for an efficient extraction method, and the obtained polysaccharide is nontoxic in nature and bears emulsifying properties. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Fucoidans, a group of high molecular weight polysaccharides derived mainly from brown algae, are characterized by their high fucose content, degree of sulfation (DS), and intra- and interspecific structural variation. Fucoidans are increasingly recognized due to various reported bioactivities, potentially beneficial for human health. To unlock their potential use within biomedical applications, a better understanding of their structure-functional relationship is needed. To achieve this, systematic bioactivity studies based on well-defined, pure fucoidans, and the establishment of standardized, satisfactory purification protocols are required. We performed a comprehensive compositional and structural characterization of crude and ultra-purified fucoidans from three kelps: Saccharina latissima (SL), Alaria esculenta (AE) and Laminaria hyperborea (LH). Further, the complement-inhibiting activity of the purified fucoidans was assessed in a human whole blood model. The purification process led to fucoidans with higher DS and fucose and lower concentrations of other monosaccharides. Fucoidans from SL and LH resembles homofucans, while AE is a heterofucan rich in galactose with comparably lower DS. Fucoidans from SL and LH showed complement-inhibiting activity in blood and blood plasma, while no inhibition was observed for AE under the same conditions. The results emphasize the importance of high DS and possibly fucose content for fucoidans' bioactive properties., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Methylation followed by depolymerization and gas chromatography (GC) is an effective methodology for the linkage analysis of polysaccharides, including fucoidan, a sulphated algal polysaccharide. However, this sample material demands attention to experimental details to prevent aberrations in the analytical result. The use of deficient bases for methylation, the presence of water, analyte degradation during hydrolysis, and coelution of the target analytes during gas chromatography create doubts about published results. We therefore investigated critical parameters of the method and carefully optimized the steps of the protocol to ensure the integrity of the results for the fucose monomers. Fucoidan from Cladosiphon okamuranus was used as reference sample to determine the glycosidic bonds, and sulphate positions in the monomer. Fucoidan in protonated form was methylated in a strictly water-free environment using lithium dimsyl as base and methyl iodide for methylation. The methylated polymer was isolated by solid phase extraction, which was crucial to recover also the highly sulfated fraction. Hydrolysis was conducted with trifluoroacetic acid. To separate all target analytes in GC-FID/MS, a stationary phase with high cyanopropyl content (HP-88) was required, as the commonly employed phenyl siloxane phases result in co-elution, which distorts the result severely., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

This study introduces a novel approach to analyze glycosidic linkages in unfractionated polysaccharides from alcohol-insoluble residues (AIRs) of five brown seaweed species. GC-MS analysis of partially methylated alditol acetates (PMAAs) enables monitoring and comparison of structural variations across different species, harvest years, and tissues with and without blanching treatments. The method detects a wide array of fucose linkages, highlighting the structural diversity in glycosidic linkages and sulfation position in fucose-containing sulfated polysaccharides. Additionally, this technique enhances cellulose quantitation, overcoming the limitations of traditional monosaccharide composition analysis that typically underestimates cellulose abundance due to incomplete hydrolysis of crystalline cellulose. The introduction of a weak methanolysis-sodium borodeuteride reduction pretreatment allows for the detection and quantitation of uronic acid linkages in alginates.

Background: α-l-Fucose confers unique functions for fucose-containing biomolecules such as human milk oligosaccharides. α-l-Fucosidases can serve as desirable tools in the application of fucosylated saccharides. Discovering novel α-l-fucosidases and elucidating their enzyme properties are always worthy tasks., Results: A GH95 family α-l-fucosidase named Afc95A_Wf was cloned from the genome of the marine bacterium Wenyingzhuangia fucanilytica and expressed in Escherichia coli. It exhibited maximum activity at 40 °C and pH 7.5. Afc95A_Wf defined a different substrate specificity among reported α-l-fucosidases, which was capable of hydrolyzing α-fucoside in CNP-fucose, Fucα1-2Galβ1-4Glc and Galβ1-4(Fucα1-3)Glc, and showed a preference for α1,2-fucosidic linkage. It adopted Asp residue in the amino acid sequence at position 391, which was distinct from the previously acknowledged residue of Asn. The predicted tertiary structure and site-directed mutagenesis revealed that Asp391 participates in the catalysis of Afc95A_Wf. The differences in the substrate specificity and catalytic site shed light on that Afc95A_Wf adopted a novel mechanism in catalysis., Conclusion: A GH95 family α-l-fucosidase (Afc95A_Wf) was cloned and expressed. It showed a cleavage preference for α1,2-fucosidic linkage to α1,3-fucosidic linkage. Afc95A_Wf demonstrated a different substrate specificity and a residue at an important catalytic site compared with known GH95 family proteins, which revealed the occurrence of diversity on catalytic mechanisms in the GH95 family. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Core fucosylation, the attachment of an α-1,6-linked-fucose to the N-glycan core pentasaccharide, is an abundant protein modification that plays critical roles in various biological processes such as cell signaling, B cell development, antibody-dependent cellular cytotoxicity, and oncogenesis. However, the tools currently used to detect core fucosylation suffer from poor specificity, exhibiting cross-reactivity against all types of fucosylation. Herein we report the development of a new chemoenzymatic strategy for the rapid and selective detection of core fucosylated glycans. This approach employs a galactosyltransferase enzyme identified from Caenorhabditis elegans that specifically transfers an azido-appended galactose residue onto core fucose via a β-1,4 glycosidic linkage. We demonstrate that the approach exhibits superior specificity toward core fucose on a variety of complex N-glycans. The method enables detection of core fucosylated glycoproteins from complex cell lysates, as well as on live cell surfaces, and it can be integrated into a diagnostic platform to profile protein-specific core fucosylation levels. This chemoenzymatic labeling approach offers a new strategy for the identification of disease biomarkers and will allow researchers to further characterize the fundamental role of this important glycan in normal and disease physiology.

Fucosylated carbohydrate antigens play critical roles in physiology and pathology with function linked to their structural details. However, the separation and structural characterization of isomeric fucosylated epitopes remain challenging analytically. Here, we report for the first time the influence of alkali metal cations (Li + , Na + , K + , Rb + , and Cs + ) and halogen anions (Cl - , Br - , and I - ) on the gas-phase conformational landscapes of common fucosylated trisaccharides (Lewis A, X, and H types 1 and 2) and tetrasaccharides (Lewis B and Y) using trapped ion mobility spectrometry coupled to mass spectrometry and theoretical calculations. Inspection of the mobility profiles of individual standards showed a dependence on the number of mobility bands with the oligosaccharide and the alkali metal and halogen; collision cross sections are reported for all of the observed species. Results showed that trisaccharides (Lewis A, X, and H types 1 and 2) can be best mobility resolved in the positive mode using the [M + Li] + molecular ion form (baseline resolution r ≈ 2.88 between Lewis X and A); tetrasaccharides can be best mobility resolved in the negative mode using the [M + I] - molecular ion form (baseline separation r ≈ 1.35 between Lewis B and Y). The correlation between the number of oligosaccharide conformers as a function of the molecular ion adduct was studied using density functional theory. Theoretical calculations revealed that smaller cations can form more stable structures based on the number of coordinations, while larger cations induced greater oligosaccharide reorganizations; candidate structures are proposed to better understand the gas-phase oligosaccharide rearrangement trends. Inspection of the candidate structures suggests that the interplay between ion size/charge density and molecular structure dictated the conformational preferences and, consequently, the number of mobility bands and the mobility separation across isomers. This work provides a fundamental understanding of the gas-phase structural dynamics of fucosylated oligosaccharides and their interaction with alkali metals and halogens.

Liposomal formulations of antibiotics for inhalation offer the potential for the delivery of high drug doses, controlled drug release kinetics in the lung, and an excellent safety profile. In this study, we evaluated the in vivo performance of a liposomal formulation for the poorly soluble, antituberculosis agent, bedaquiline. Bedaquiline was encapsulated within monodisperse liposomes of ∼70 nm at a relatively high drug concentration (∼3.6 mg/mL). Formulations with or without fucose residues, which bind to C-type lectin receptors and mediate a preferential binding to macrophage mannose receptor, were prepared, and efficacy was assessed in an in vivo C3HeB/FeJ mouse model of tuberculosis infection (H37Rv strain). Seven intranasal instillations of 5 mg/kg bedaquiline formulations administered every second day resulted in a significant reduction in lung burden (∼0.4-0.6 Δlog 10 CFU), although no differences between fucosylated and nonfucosylated formulations were observed. A pharmacokinetic study in healthy, noninfected Balb/c mice demonstrated that intranasal administration of a single dose of 2.5 mg/kg bedaquiline liposomal formulation (fucosylated) improved the lung bioavailability 6-fold compared to intravenous administration of the same formulation at the same dose. Importantly, intranasal administration reduced systemic concentrations of the primary metabolite, N -desmethyl-bedaquiline (M2), compared with both intravenous and oral administration. This is a clinically relevant finding as the M2 metabolite is associated with a higher risk of QT-prolongation in predisposed patients. The results clearly demonstrate that a bedaquiline liposomal inhalation suspension may show enhanced antitubercular activity in the lung while reducing systemic side effects, thus meriting further nonclinical investigation.

Bovine intestinal alkaline phosphatase (biALP), a membrane-bound plasma metalloenzyme, maintains intestinal homeostasis, regulates duodenal surface pH, and protects against infections caused by pathogenic bacteria. The N-glycans of biALP regulate its enzymatic activity, protein folding, and thermostability, but their structures are not fully reported. In this study, the structures and quantities of the N-glycans of biALP were analyzed by liquid chromatography-electrospray ionization-high energy collision dissociation-tandem mass spectrometry. In total, 48 N-glycans were identified and quantified, comprising high-mannose [6 N-glycans, 33.1 % (sum of relative quantities of each N-glycan)], hybrid (6, 11.9 %), and complex (36, 55.0 %) structures [bi- (13, 26.1 %), tri- (16, 21.5 %), and tetra-antennary (7, 7.4 %)]. These included bisecting N-acetylglucosamine (33, 56.6 %), mono-to tri-fucosylation (32, 53.3 %), mono-to tri-α-galactosylation (16, 20.7 %), and mono-to tetra-β-galactosylation (36, 58.5 %). No sialylation was identified. N-glycans with non-bisecting GlcNAc (9, 10.3 %), non-fucosylation (10, 13.6 %), non-α-galactosylation (26, 46.2 %), and non-β-galactosylation (6, 8.4 %) were also identified. The activity (100 %) of biALP was reduced to 37.3 ± 0.2 % (by de-fucosylation), 32.7 ± 2.9 % (by de-α-galactosylation), and 0.2 ± 0.2 % (by de-β-galactosylation), comparable to inhibition by 10 -4 to 10 1  mM EDTA, a biALP inhibitor. These results indicate that fucosylated and galactosylated N-glycans, especially β-galactosylation, affected the activity of biALP. This study is the first to identify 48 diverse N-glycan structures and quantities of bovine as well as human intestinal ALP and to demonstrate the importance of the role of fucosylation and galactosylation for maintaining the activity of biALP., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The frequent mutations of influenza A virus (IAV) have led to an urgent need for the development of innovative antiviral drugs. Glycopolymers offer significant advantages in biomedical applications owing to their biocompatibility and structural diversity. However, the primary challenge lies in the design and synthesis of well-defined glycopolymers to precisely control their biological functionalities. In this study, functional glycopolymers with sulfated fucose and 6'-sialyllactose were successfully synthesized through ring-opening metathesis polymerization and a postmodification strategy. The optimized heteropolymer exhibited simultaneous targeting of hemagglutinin and neuraminidase on the surface of IAV, as evidenced by MU-NANA assay and hemagglutination inhibition data. Antiviral experiments demonstrated that the glycopolymer displayed broad and efficient inhibitory activity against wild-type and mutant strains of H1N1 and H3N2 subtypes in vitro , thereby establishing its potential as a dual-targeted inhibitor for combating IAV resistance.

Alpha-1-acid glycoprotein (AGP) is a heterogeneous glycoprotein fulfilling key roles in many biological processes, including transport of drugs and hormones and modulation of inflammatory and immune responses. The glycoform profile of AGP is known to change depending on (patho)physiological states such as inflammatory diseases or pregnancy. Besides complexity originating from five N-glycosylation sites, the heterogeneity of the AGP further expands to genetic variants. To allow in-depth characterization of this intriguing protein, we developed a method using anion exchange chromatography (AEX) coupled to mass spectrometry (MS) revealing the presence of over 400 proteoforms differing in their glycosylation or genetic variants. More precisely, we could determine that AGP mainly consists of highly sialylated higher antennary structures with on average 16 sialic acids and 0 or 1 fucose per protein. Interestingly, a slightly higher level of fucosylation was observed for AGP1 variants compared to that of AGP2. Proteoform assignment was supported by integrating data from complementary MS-based approaches, including AEX-MS of an exoglycosidase-treated sample and glycopeptide analysis after tryptic digestion. The developed analytical method was applied to characterize AGP from plasma of women during and after pregnancy, revealing differences in glycosylation profiles, specifically in the number of antennae, HexHexNAc units, and sialic acids.

Dysregulation of protein core-fucosylation plays a pivotal role in the onset, progression, and immunosuppression of cancer. However, analyzing core-fucosylation, especially the accurate determination of the core-fucosylation (CF) site occupancy ratio, remains challenging. To address these problems, we developed a truncation strategy that efficiently converts intact glycopeptides with hundreds of different glycans into two truncated forms, i.e., a monosaccharide HexNAc and a disaccharide HexNAc+core-fucose. Further combination with data-independent analysis to form an integrated platform allowed the measurement of site-specific core-fucosylation abundances and the determination of the CF occupancy ratio with high reproducibility. Notably, three times CF sites were identified using this strategy compared to conventional methods based on intact glycopeptides. Application of this platform to characterize protein core-fucosylation in two breast cancer cell lines, i.e., MDA-MB-231 and MCF7, yields a total of 1615 unique glycosites and about 900 CF sites from one single LC-MS/MS analysis. Differential analysis unraveled the distinct glycosylation pattern for over 201 cell surface drug targets between breast cancer subtypes and provides insights into developing new therapeutic strategies to aid precision medicine. Given the robust performance of this platform, it would have broad application in discovering novel biomarkers based on the CF glycosylation pattern, investigating cancer mechanisms, as well as detecting new intervention targets.

N-glycosylation of the Fc part is a (critical) quality attribute of therapeutic antibodies and Fc-containing biotherapeutics, that impacts their stability, immunogenicity, pharmacokinetics, and effector functions. Current glycosylation analysis methods focus on the absolute amounts of glycans, neglecting the apparent glycan distribution over the entirety of proteins. The combination of the two Fc N-glycans, herein referred to as glyco-pair, therefore remains unknown, which is a major drawback for N-glycan impact assessment. This study presents a comprehensive workflow for the analysis and characterization of Fc N-glycan pairing in biotherapeutics, addressing the limitations of current glycosylation analysis methods. The applicability of the method across various biotherapeutic proteins including antibodies, bispecific antibody formats, and a Fc-Fusion protein is demonstrated, and the impact of method conditions on glycan pairing analysis is highlighted. Moreover, the influence of the molecular format, Fc backbone, production process, and cell line on glycan pairing pattern was investigated. The results underscore the significance of comprehensive glycan pairing analysis to accurately assess the impact of N-glycans on important product quality attributes of therapeutic antibodies and Fc-containing biotherapeutics., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Maximilian Meudt, Julia Baumeister, Michaela Blech and Fabian Higel are employees of Boehringer Ingelheim GmbH & Co. KG, which is developing, manufacturing, and marketing biopharmaceutical products., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Five GH29B α-1,3/4-l-fucosidases (EC 3.2.1.111) were investigated for their ability to catalyze the formation of the human milk oligosaccharide lacto- N -fucopentaose II (LNFP II) from lacto- N -tetraose (LNT) and 3-fucosyllactose (3FL) via transglycosylation. We studied the effect of pH on transfucosylation and hydrolysis and explored the impact of specific mutations using molecular dynamics simulations. LNFP II yields of 91 and 65% were obtained for the wild-type SpGH29 C and CpAfc2 enzymes, respectively, being the highest LNFP II transglycosylation yields reported to date. BbAfcB and BiAfcB are highly hydrolytic enzymes. The results indicate that the effects of pH and buffer systems are enzyme-dependent yet relevant to consider when designing transglycosylation reactions. Replacing Thr284 in BiAfcB with Val resulted in increased transglycosylation yields, while the opposite replacement of Val258 in SpGH29 C and Val289 CpAfc2 with Thr decreased the transfucosylation, confirming a role of Thr and Val in controlling the flexibility of the acid/base loop in the enzymes, which in turn affects transglycosylation. The substitution of an Ala residue with His almost abolished secondary hydrolysis in CpAfc2 and BbAfcB. The results are directly applicable in the enhancement of transglycosylation and may have significant implications for manufacturing of LNFP II as a new infant formula ingredient.

Fucosylation is an important quality attribute for therapeutic antibodies. Afucosylated antibodies exhibit higher therapeutic efficacies than their fucosylated counterparts through antibody-dependent cellular cytotoxicity (ADCC) mechanism. Since higher potency is beneficial in reducing dose or duration of the treatment, afucosylated antibodies have attracted a great deal of interest in biotherapeutics development. In this study, novel small molecules GDP-D-Rhamnose and its derivatives (Ac-GDP-D-Rhamnose and rhamnose sodium phosphate) were synthesized to inhibit the enzyme in the GDP-fucose synthesis pathway. Addition of these compounds into cell culture increased antibody afucosylation levels in a dose-dependent manner and had no significant impact on other protein quality attributes. A novel and effective mechanism to generate afucosylated antibody is demonstrated for biologics discovery, analytical method development, process development, and other applications., (© 2024 American Institute of Chemical Engineers.)

Fucosylated chondroitin sulfate is a unique glycosaminoglycan isolated from sea cucumbers, with excellent anticoagulant activity. The fucosyl branch in FCS is generally located at the 3-OH of D -glucuronic acid but, recently, a novel structure with α- L -fucose linked to the 6-OH of N -acetyl-galactosamine has been found. Here, using functionalized monosaccharide building blocks, we prepared novel FCS tetrasaccharides with fucosyl branches both at the 6-OH of GalNAc and 3-OH of GlcA. In the synthesis, the protective group strategy of selective O -sulfation, as well as stereoselective glycosylation, was established, which enabled the efficient synthesis of the specific tetrasaccharide compounds. This research enriches knowledge on the structural types of FCS oligosaccharides and facilitates the exploration of the structure-activity relationship in the future.

Fucosylation is an important structural feature of glycans and plays an essential role in the regulation of glycoprotein functions. Fucosylation can be classified into core- (CF) and antenna-fucosylation (AF, also known as (sialyl-) Lewis) based on the location on N -glycans, and they perform distinct biological functions. In this study, core- and antenna-fucosylated N -glycans on human serum glycoproteins that hold great clinical application values were systematically characterized at the site-specific level using StrucGP combined with the recently developed fucosylation assignment method. The results showed that fucosylation was widely distributed on serum glycoproteins, with 50% of fucosylated glycopeptides modified by AF N -glycans, 37% by CF N -glycans, and 13% by dual-fucosylated N -glycans. Interestingly, CF and AF N -glycans preferred to modify different groups of serum glycoproteins with different tissue origins and were involved in distinctive biological processes. Specifically, AF N -glycoproteins are mainly from the liver and participated in complement activation, blood coagulation, and endopeptidase activities, while CF N -glycoproteins originate from diverse tissues and are mainly involved in cell adhesion and signaling transduction. These data further enhanced our understanding of fucosylation on circulation glycoproteins.

Lipopolysaccharides (LPSs) are major components of the outer membranes of Gram-negative bacteria. In this work, the structure of the O-polysaccharide of Ochrobactrum quorumnocens T1Kr02 was identified by nuclear magnetic resonance (NMR), and the physical-chemical properties and biological activity of LPS were also investigated. The NMR analysis showed that the O-polysaccharide has the following structure: →2)-β-d-Fuc f -(1→3)-β-d-Fuc p -(1→. The structure of the periplasmic glucan coextracted with LPS was established by NMR spectroscopy and chemical methods: →2)-β-d-Glc p -(1→. Non-stoichiometric modifications were identified in both polysaccharides: 50% of d-fucofuranose residues at position 3 were O-acetylated, and 15% of d-Glc p residues at position 6 were linked with succinate. This is the first report of a polysaccharide containing both d-fucopyranose and d-fucofuranose residues. The fatty acid analysis of the LPS showed the prevalence of 3-hydroxytetradecanoic, hexadecenoic, octadecenoic, lactobacillic, and 27-hydroxyoctacosanoic acids. The dynamic light scattering demonstrated that LPS (in an aqueous solution) formed supramolecular particles with a size of 72.2 nm and a zeta-potential of -21.5 mV. The LPS solution (10 mkg/mL) promoted the growth of potato microplants under in vitro conditions. Thus, LPS of O. quorumnocens T1Kr02 can be recommended as a promoter for plants and as a source of biotechnological production of d-fucose.

l-fucose is a six-carbon sugar that has potential applications in many fields. It exerts antitumor effects and could relieve intestinal disease. It exhibits potential as an emulsifier in the food industry. It is also used as a functional food and in anti-aging skincare products. However, at present, it is not possible to prepare high-purity l-fucose on a large scale, and its preparation needs further development. This review summarizes the preparation methods of l-fucose including chemical synthesis, enzymatic synthesis, microbial fermentation, and separation and purification from algae. The detection methods of l-fucose are also introduced in detail, such as l-fucose-specific lectin, detection l-fucose dehydrogenase, cysteine-sulfuric acid method, high-performance liquid chromatography, gas chromatography, and biosensors. In this review, the properties and pharmacological effects of l-fucose; preparation methods, and the commonly used detection methods of l-fucose are reviewed to serve as a reference material., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

PhoSL (Pholiota squarrosa Lectin) has an exceptional binding affinity for biomolecules with core-fucosylated N-glycans. This modification involves the addition of fucose to the inner N-acetylglucosamine within the N-glycan structure and is known to influence many physiological processes. Nevertheless, the molecular interactions underlying high-affinity binding of native PhoSL to core-fucosylated N-glycans remain largely unknown. In this study, we devised a strategy to produce PhoSL with the essential structural characteristics of the native protein (n-PhoSL). To do so, a fusion protein was expressed in E. coli and purified. Then, enzymatic cleavage and incubation with glutathione were utilized to recapitulate the native primary structure and disulfide bonding pattern. Subsequently, we identified the residues crucial for n-PhoSL binding to core-fucosylated chitobiose (N2F) via NMR spectroscopy. Additionally, crystal structures were solved for both apo n-PhoSL and its N2F complex. These analyses suggested a pivotal role of the N-terminal amine in maintaining the integrity of the binding pocket and actively contributing to core-fucose recognition. In support of this idea, the inclusion of additional residues at the N-terminus considerably reduced binding affinity and PhoSL cytotoxicity toward breast cancer cells. Taken together, these findings can facilitate the utilization of PhoSL in basic research, diagnostics and therapeutic strategies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Fucosylation is facilitated by converting GDP-mannose to GDP-4-keto-6-deoxymannose, which GDP-mannose 4,6-dehydratase, a crucial enzyme in the route, carries out. One of the most prevalent glycosylation alterations linked to cancer has reportedly been identified as fucosylation. There is mounting evidence that GMDS is intimately linked to the onset and spread of cancer. Furthermore, the significance of long-chain non-coding RNAs in the development and metastasis of cancer is becoming more well-recognized, and the regulatory mechanism of lncRNAs has emerged as a prominent area of study in the biological sciences. GMDS-AS1, an antisense RNA of GMDS, was discovered to have the potential to be an oncogene. We have acquired and analyzed relevant data to understand better how GMDS-AS1 and its lncRNA work physiologically and in tumorigenesis and progression. Additionally, we have looked into the possible effects of these molecules on cancer treatment approaches and patient outcomes. The physiological roles and putative processes of GMDS and lncRNA GMDS-AS1 throughout the development and progression of tumors have been assembled and examined. We also examined how these chemicals might affect patient prognosis and cancer therapy approaches. GMDS and GMDS-AS1 were determined to be research subjects by searching and gathering pertinent studies using the PubMed system. The analysis of these research articles demonstrated the close relationship between GMDS and GMDS-AS1 and tumorigenesis and the factors that influence them. GMDS plays a vital role in regulating fucosylation. The related antisense gene GMDS-AS1 affects the biological behaviors of cancer cells through multiple pathways, including the key processes of proliferation, migration, invasion, and apoptosis, providing potential biomarkers and therapeutic targets for cancer treatment and prognosis assessment., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Human milk oligosaccharides (HMOs) have attracted considerable attention owing to their unique physiological functions. Two important tetrasaccharides, lacto- N -tetraose (LNT) and lacto- N -neotetraose (LNnT), are core structures of HMOs. Their safety has been evaluated and they can be added to infant formula as functional ingredients. The fucosylated derivatives of LNT and LNnT, mainly lacto- N -fucopentaose (LNFP) I, LNFP II, LNFP III, and lacto- N -difucohexaose I, exhibit prominent physiological characteristics, including modificating the intestinal microbiota, immunomodulation, anti-bacterial activities, and antiviral infection. However, they have received lesser attention than 2'-fucosyllactose. As precursors, LNT and LNnT are connected to one or two fucosyl units through α1,2/3/4 glycosidic bonds, forming a series of compounds with complex structures. These complex fucosylated oligosaccharides can be biologically synthesized using enzymatic and cell factory approaches. This review summarizes the occurrence, physiological effects, and biosynthesis of fucosylated LNT and LNnT derivatives and their future development.

Lacto-N-fucopentaose I (LNFP I) is an abundant and important fucosylated human milk oligosaccharide (HMO). Here, an efficient LNFP I-producing strain without by-product 2'-fucosyllactose (2'-FL) was developed by advisable stepwise de novo pathway construction in Escherichia coli. Specifically, the genetically stable lacto-N-triose II (LNTri II)-producing strains were constructed by the multicopy integration of β1,3-N-acetylglucosaminyltransferase. LNTri II can be further converted to lacto-N-tetraose (LNT) by LNT-producing β1,3-galactosyltransferase. The de novo and salvage pathways of GDP-fucose were introduced into highly efficient LNT-producing chassis. Specific α1,2-fucosyltransferase was verified to eliminate by-product 2'-FL, and binding free energy of the complex was analyzed to explain the product distribution. Subsequently, further attempts aiming to improve α1,2-fucosyltransferase activity and the supply of GDP-fucose were carried out. Our engineering strategies enabled the stepwise de novo construction of strains that produced up to 30.47 g/L of extracellular LNFP I, without accumulation of 2'-FL, and with only minor intermediates residue., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The structural and functional relationships of glycosaminoglycans (GAGs) derived from marine organisms have been investigated, suggesting that marine invertebrates, particularly Bivalvia, are abundant sources of highly sulfated or branched GAGs. In this study, we identified a novel fucosylated heparan sulfate (Fuc-HS) from the midgut gland of the Japanese scallop, Patinopecten yessoensis. Scallop HS showed resistance to GAG-degrading enzymes, including chondroitinases and heparinases, and susceptibility to heparinases increased when scallop HS was treated with mild acid hydrolysis, which removes the fucosyl group. Moreover, 1 H NMR detected significant signals near 1.2-1.3 ppm corresponding to the H-6 methyl proton of fucose residues and small H-3 (3.59 ppm) or H-2 (3.39 ppm) signals of glucuronate (GlcA) were detected, suggesting that the fucose moiety is attached to the C-3 position of GlcA in scallop HS. GC-MS detected peaks corresponding to 1, 3, 5-tri-O-acetyl-2, 4-di-O-methyl- L -fucitol and 1, 4, 5-tri-O-acetyl-2, 3-di-O-methyl- L -fucitol, suggesting that the fucose moiety is 3-O- or 4-O-sulfated. Furthermore, scallop HS showed anti-coagulant and neurite outgrowth-promoting (NOP) activities. These results suggest that the midgut gland of scallops is a valuable source of Fuc-HS with biological activities., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The majority of nonbacterial gastroenteritis in humans and livestock is caused by noroviruses. Like most RNA viruses, frequent mutations result in various norovirus variants. The strain-dependent binding profiles of noroviruses to fucose are supposed to facilitate norovirus infection. It remains unclear, however, what the molecular mechanism behind strain-dependent functioning is. In this study, by applying atomic force microscopy (AFM) nanoindentation technology, we studied norovirus-like particles (noroVLPs) of three distinct human norovirus variants. We found differences in viral mechanical properties even between the norovirus variants from the same genogroup. The noroVLPs were then subjected to fucose treatment. Surprisingly, after fucose treatment, the previously found considerable differences in viral mechanical properties among these variants were diminished. We attribute a dynamic switch of the norovirus P domain upon fucose binding to the reduced differences in viral mechanical properties across the tested norovirus variants. These findings shed light on the mechanisms used by norovirus capsids to adapt to environmental changes and, possibly, increase cell infection. Hereby, a new step towards connecting viral mechanical properties to viral prevalence is taken.

Fucose is a signaling carbohydrate that is attached at the end of glycan processing. It is involved in a range of processes, such as the selectin-dependent leukocyte adhesion or pathogen-receptor interactions. Mass-spectrometric techniques, which are commonly used to determine the structure of glycans, frequently show fucose-containing chimeric fragments that obfuscate the analysis. The rearrangement leading to these fragments-often referred to as fucose migration-has been known for more than 25 years, but the chemical identity of the rearrangement product remains unclear. In this work, we combine ion-mobility spectrometry, radical-directed dissociation mass spectrometry, cryogenic IR spectroscopy of ions, and density-functional theory calculations to deduce the product of the rearrangement in the model trisaccharides Lewis x and blood group H2. The structural search yields the fucose moiety attached to the galactose with an α(1→6) glycosidic bond as the most likely product., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Glycoside hydrolase family 29 (GH29) encompasses α-L-fucosidases, i.e. enzymes that catalyze the hydrolytic release of fucose from fucosylated glycans, including N- and O-linked glycans on proteins, and these α-L-fucosidases clearly play important roles in biology. GH29 enzymes work via a retaining exo-action mechanism, and some can catalyze transfucosylation. There is no formal subfamily division of GH29 α-L-fucosidases, but they are nonetheless divided into two subfamilies: GH29A having a range of substrate specificities and GH29B having narrower substrate specificity. However, the sequence traits that determine the substrate specificity and transglycosylation ability of GH29 enzymes are not well characterized. Here, we present a new functional map of family GH29 members based on peptide-motif clustering via CUPP (conserved unique peptide patterns) and compare the substrate specificity and transglycosylation activity of 21 representative α-L-fucosidases across the 53 CUPP groups identified. The 21 enzymes exhibited different enzymatic rates on 8 test substrates, CNP-Fuc, 2'FL, 3FL, Lewisa, Lewisx, Fuc-α1,6-GlcNAc, Fuc-α1,3-GlcNAc, and Fuc-α1,4-GlcNAc. Certain CUPP groups clearly harbored a particular type of enzymes, e.g. the majority of the enzymes having activity on Lewisa or Lewisx categorized in the same CUPP clusters. In general, CUPP was useful for resolving GH29 into functional diversity subgroups when considering hydrolytic activity. In contrast, the transglycosylation capacity of GH29 α-L-fucosidases was distributed across a range of CUPP groups. Transglycosylation thus appears to be a common trait among these enzymes and not readily predicted from sequence comparison., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Fucose is a monosaccharide commonly found in mammalian, insect, microbial and plant glycans. The removal of terminal α-l-fucosyl residues from oligosaccharides and glycoconjugates is catalysed by α-l-fucosidases. To date, glycoside hydrolases (GHs) with exo-fucosidase activity on α-l-fucosylated substrates (EC 3.2.1.51, EC 3.2.1.-) have been reported in the GH29, GH95, GH139, GH141 and GH151 families of the Carbohydrate Active Enzymes (CAZy) database. Microbes generally encode several fucosidases in their genomes, often from more than one GH family, reflecting the high diversity of naturally occuring fucosylated structures they encounter. Functionally characterised microbial α-l-fucosidases have been shown to act on a range of substrates with α-1,2, α-1,3, α-1,4 or α-1,6 fucosylated linkages depending on the GH family and microorganism. Fucosidases show a modular organisation with catalytic domains of GH29 and GH151 displaying a (β/α)8-barrel fold while GH95 and GH141 show a (α/α)6 barrel and parallel β-helix fold, respectively. A number of crystal structures have been solved in complex with ligands, providing structural basis for their substrate specificity. Fucosidases can also be used in transglycosylation reactions to synthesise oligosaccharides. This mini review provides an overview of the enzymatic and structural properties of microbial α-l-fucosidases and some insights into their biological function and biotechnological applications., (© 2023 The Author(s).)

Fucose is a ubiquitous monosaccharide associated to major classes of glycans. A main obstacle to the sequencing of fucosylated glycans is the migration of fucose, which leads to misinterpretations in mass spectrometry analysis. Here, using ion vibrational spectroscopy, we resolve the structure of fucosylated fragments of Lewis and blood group H antigen trisaccharides and we unveil the position and linkage of the fucose after migration. Our findings demonstrate that the structure of fragment ions resulting from fucose migration can be characterized. Additionally, we report a new type of fucose migration, which does not feature any change of mass and therefore had not been previously reported: it consists of a local migration where the fucose changes its position remaining on the initial residue. Our approach allows the characterization of glycans, an essential step to interpret glycomics data, as well as to understand underlying processes at play in mass spectrometry., (© 2023 Wiley-VCH GmbH.)

Biologically active phytochemicals from pumpkin reveal versatile medical applications, though little is known about their antiviral activity. The fucose-rich polysaccharide extraction conditions were optimized through Box-Behnken design and purified by column chromatography. The purified fucose-rich polysaccharide was characterized through SEM, FT-IR, 1 H NMR, XRD, TGA, and GS-MS. The analysis results revealed an irregular and porous surface of the purified polysaccharide with high fucose, rhamnose, galactose, and glucose contents. The tested fucose-rich polysaccharides revealed significant antioxidant and anti-inflammatory activity at very low concentrations. The purified fucose-rich polysaccharides exerted a broad-spectrum antiviral activity against both DNA and RNA viruses, accompanied by high safety toward normal cells, where the maximum safe doses (EC 100 ) were estimated to be about 3-3.9 mg/mL for both Vero and PBMC cell lines. Treatment of HCV, ADV7, HSV1, and HIV viruses with the purified polysaccharides showed a potent dose-dependent inhibitory activity with IC 50 values of 95.475, 20.96, 5.213, and 461.75 μg/mL, respectively. This activity was hypothesized to be through inhibiting the viral entry in HCV infection and inhibiting the reverse transcriptase activity in HIV. The current study firstly reported the antioxidant, anti-inflammatory, and antiviral activities of Cucurbita maxima fucose-rich polysaccharide against several viral infections., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2023. Published by Elsevier B.V.)

Brown macroalgae are an important source of polysaccharides, mainly fucose-containing sulphated polysaccharides (FCSPs), associated with several biological activities. However, the structural diversity and structure-function relationships for their bioactivities are still undisclosed. Thus, the aim of this work was to characterize the chemical structure of water-soluble Saccharina latissima polysaccharides and evaluate their immunostimulatory and hypocholesterolemic activities, helping to pinpoint a structure-activity relationship. Alginate, laminarans (F1, neutral glucose-rich polysaccharides), and two fractions (F2 and F3) of FCSPs (negatively charged) were studied. Whereas F2 is rich in uronic acids (45 mol%) and fucose (29 mol%), F3 is rich in fucose (59 mol%) and galactose (21 mol%). These two fractions of FCSPs showed immunostimulatory activity on B lymphocytes, which could be associated with the presence of sulphate groups. Only F2 exhibited a significant effect in reductions in in vitro cholesterol's bioaccessibility attributed to the sequestration of bile salts. Therefore, S. latissima FCSPs were shown to have potential as immunostimulatory and hypocholesterolemic functional ingredients, where their content in uronic acids and sulphation seem to be relevant for the bioactive and healthy properties.

The d-Glc N Ac moiety in sialyl Lewis x (sLe x , 1 ) acts predominantly as a linker to position the d-Gal and the l-Fuc moieties in the bioactive spatial orientation. The hypothesis has been made that the NHAc group of Glc N Ac pushes the fucose underneath the galactose and, thus, contributes to the stabilization of the bioactive conformation of the core of sLe x ( 1 ). To test this hypothesis, Glc N Ac mimetics consisting of ( R , R )-1,2-cyclohexanediols substituted with alkyl and aryl substituents adjacent to the linking position of the fucose moiety were synthesized. To explore a broad range of extended and spatially demanding R-groups, an enzymatic approach for the synthesis of 3-alkyl/aryl-1,2-cyclohexanediols ( 3b-n ) was applied. These cyclohexanediol derivatives were incorporated into the sLe x mimetics 2b-n . For analyzing the relationship of affinity and core conformation, a 1 H NMR structural-reporter-group concept was applied. Thus, the chemical shift of H-C5 Fuc proved to be a sensitive indicator for the degree of pre-organization of the core of this class of sLe x mimetics and therefore could be used to quantify the contribution of the R-groups.

Fucoidans are fucose rich sulfated polysaccharides that are found in the cell wall of brown seaweeds and have been shown to have several beneficial bioactivities. In the present study, we report a new enzymatic extraction technique for the production of pure and intact fucoidans from the two brown seaweeds Saccharina latissima and Alaria esculenta. This new extraction protocol uses the commercial cellulase blend Cellic® CTec2 in combination with endo- and exo-acting thermophilic alginate lyases. The fucoidans obtained by this extraction technique are compared to traditionally extracted fucoidans in terms of chemical compositions and molecular weights and are shown to contain significantly higher amounts of fucose and sulfate, the main components of fucoidans, while cellulose, laminarin, and alginate contamination is low. Thus, by using this combination of enzymes, the extracted fucoidans do not undergo depolymerization during extraction and additional purification steps are not needed. The high purity fucoidans isolated by this new enzymatic extraction technique can be used to provide insight into the different fucoidan structures and biological activities., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

The inhibition of carbohydrate-lectin interactions is being explored as an efficient approach to anti adhesion therapy and biofilm destabilization, two alternative antimicrobial strategies that are being explored against resistant pathogens. BC2L-C is a new type of lectin from Burkholderia cenocepacia that binds (mammalian) fucosides at the N -terminal domain and (bacterial) mannosides at the C -terminal domain. This double carbohydrate specificity allows the lectin to crosslink host cells and bacterial cells. We have recently reported the design and generation of the first glycomimetic antagonists of BC2L-C, β- C - or β- N -fucosides that target the fucose-specific N- terminal domain (BC2L-C-Nt). The low water solubility of the designed N -fucosides prevented a full examination of this promising series of ligands. In this work, we describe the synthesis and biophysical evaluation of new L-fucosyl and L-galactosyl amides, designed to be water soluble and to interact with BC2L-C-Nt. The protein-ligand interaction was investigated by Saturation Transfer Difference NMR, Isothermal Titration Calorimetry and crystallographic studies. STD-NMR experiments showed that both fucosyl and galactosyl amides compete with α-methyl fucoside for lectin binding. A new hit compound was identified with good water solubility and an affinity for BC2L-C-Nt of 159 μM (ITC), which represents a one order of magnitude gain over α-methyl fucoside. The x-ray structure of its complex with BC2L-C-Nt was solved at 1.55 Å resolution.

Aims: l-Fuculose is a valuable rare sugar that is used to treat a variety of ailments, including HIV, cancer, Hepatitis B, human lysosomal disease (fucosidosis), and cardio-protective medications. The enzymatic approach for the production of l-fuculose using l-fucose as a substrate would be an advantageous method with a wide range of industrial applications. The objective of this study is the characterization of recombinant l-fucose isomerase from Paenibacillus rhizosphaerae (Pa-LFI) for the production of l-fuculose from an inexpensive and natural source (fucoidan) as well as its comparison with commercial l-fucose (Sigma-Aldrich)., Methods and Results: Fucoidan, a fucose-containing polysaccharide (FPs), was isolated from Undaria pinnatifida, subsequently hydrolyzed, and characterized before the enzymatic production of l-fuculose. The results elaborate that FPs contain 35.9% of fucose along with other kinds of monosaccharides. The purified Pa-LFI exhibited a single band at 65 kDa and showed it as a hexamer with a native molecular mass of 396 kDa. The highest activity of 104.5 U mg-1 of Pa-LFI was perceived at a temperature of 50°C and pH 6.5 in the presence of 1 mM of Mn2+. The Pa-LFI revealed a melting temperature (Tm) of 75°C and a half-life of 12.6 h at 50°C. It exhibited that Pa-LFI with aldose substrate (l-fucose), has a stronger isomerizing activity, disclosing Km,kcat, and kcat/Km 86.2 mM, 32 831 min-1, and 335 min-1 mM-1, respectively. After reaching equilibrium, Pa-LFI efficiently catalyzed the reaction to convert l-fucose into l-fuculose and the conversion ratios of l-fuculose from 100 g L-1 of FPs and commercial fucose were around 6% (5.6 g L-1) and 30% (30.2 g L-1), respectively., Conclusions: According to the findings of the current study, the Pa-LFI will be useful in the manufacturing of l-fuculose using an effective and easy approach that produces no by-products., (© The Author(s) 2022. Published by Oxford University Press on behalf of Applied Microbiology International.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (S protein) is highly N-glycosylated, and a "glycan shield" is formed to limit the access of other molecules; however, a small open area coincides with the interface to the host's receptor and also neutralising antibodies. Most of the variants of concern have mutations in this area, which could reduce the efficacy of existing antibodies. In contrast, N-glycosylation sites are relatively invariant, and some are essential for infection. Here, we observed that the S proteins of the ancestral (Wuhan) and Omicron strains bind with Pholiota squarrosa lectin (PhoSL), a 40-amino-acid chemically synthesised peptide specific to core-fucosylated N-glycans. The affinities were at a low nanomolar level, which were ~ 1000-fold stronger than those between PhoSL and the core-fucosylated N-glycans at the micromolar level. We demonstrated that PhoSL inhibited infection by both strains at similar submicromolar levels, suggesting its broad-spectrum effect on SARS-CoV-2 variants. Cryogenic electron microscopy revealed that PhoSL caused an aggregation of the S protein, which was likely due to the multivalence of both the trimeric PhoSL and S protein. This characteristic is likely relevant to the inhibitory mechanism. Structural modelling of the PhoSL-S protein complex indicated that PhoSL was in contact with the amino acids of the S protein, which explains the enhanced affinity with S protein and also indicates the significant potential for developing specific binders by the engineering of PhoSL., (© 2022 Federation of European Biochemical Societies.)

Accurate identification of core fucosylation on N -glycopeptides remains challenging due to fucose migration during mass spectrometry analysis. Here, we introduce a simple and straightforward method for core-fucosylated glycopeptide recognition based on the relative intensities of Y1+Fuc ions compared with their corresponding Y1 ions (labeled as Y1+Fuc/Y1 or simply Y1F/Y1 ratio > 0.1) in low-energy HCD-based spectra. The method was first developed by systematically evaluating the influence of fucose migration on the Y1F ion from antenna fucoses based on the distribution of the Y1F/Y1 ratios in the MS/MS spectra of antenna-fucosylated glycopeptides from Fut8 -/- mouse brain. The feasibility of the method was then confirmed by using two standard glycoproteins, comparison with glycopeptides in Fut8 +/+ mouse brain with/without in silico core-fucosylation removal, and Y1F/Y1 ratio alterations under a lower HCD energy. This method will be applicable to the manual interpretation and software-based high-throughput analysis of core-fucosylated glycopeptides.

Fungal and bacterial pathogens causing lung infections often use lectins to mediate adhesion to glycoconjugates at the surface of host tissues. Given the rapid emergence of resistance to the treatments in current use, β-propeller lectins such as FleA from Aspergillus fumigatus , SapL1 from Scedosporium apiospermum, and BambL from Burkholderia ambifaria have become appealing targets for the design of anti-adhesive agents. In search of novel and cheap anti-infectious agents, we synthesized multivalent compounds that can display up to 20 units of fucose, the natural ligand. We obtained nanomolar inhibitors that are several orders of magnitude stronger than their monovalent analogue according to several biophysical techniques (i.e., fluorescence polarization, isothermal titration calorimetry, and bio-layer interferometry). The reason for high affinity might be attributed to a strong aggregating mechanism, which was examined by analytical ultracentrifugation. Notably, the fucosylated inhibitors reduced the adhesion of A. fumigatus spores to lung epithelial cells when administered 1 h before or after the infection of human lung epithelial cells. For this reason, we propose them as promising anti-adhesive drugs for the prevention and treatment of aspergillosis and related microbial lung infections.

Stichopus monotuberculatus is a tropical sea cucumber species and used as a folk medicine and tonic food. In this study, a fucosylated glycosaminoglycan (SmFG), the depolymerized SmFG (dSmFG) and its oligosaccharide fractions were prepared. The SmFG and its depolymerized products were comprised of a chondroitin-sulfate-E backbone, and various sulfated fucose side chains, including an unusual disaccharide side chain connected to the C-3 position of D-glucuronic acid (GlcA) or GlcA-ol. A peeling reaction occurred during the deaminative depolymerization process. The dSmFG and its fractions showed strong anticoagulant activity by selectively inhibiting intrinsic tenase complex, and had no anti-factor IIa, Xa and VIIa activity. The anticoagulant activity reduced with the decrease of molecular weight, and the unusual branch and novel reducing end may enhance the anticoagulant activity. These findings can provide significant information for development and utilization of depolymerized products from SmFG in food and pharmaceutical industries., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Ltd.)

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., (© 2022 Wiley-VCH GmbH.)

Functional ingredients for human health have recently become the focus of research. One such potentially versatile therapeutic component is fucose-containing sulfated polysaccharides (FCSPs), referred to as fucoidans. The exploitation of marine brown algae provides a rich source of FCSPs because of their role as a structural component of the cell wall. Fucoidans are characterized by a sulfated fucose backbone. However, the structural characterization of FCSPs is impeded by their structural diversity, molecular weight, and complexity. The extraction and purification conditions significantly influence the yield and structural alterations. Inflammation is the preliminary response to potentially injurious inducements, and it is of the utmost importance for modulation in the proper direction. Improper manipulation and/or continuous stimuli could have detrimental effects in the long run. The web of immune responses mediated through multiple modulatory/cell signaling components can be addressed through functional ingredients, benefiting patients with no side effects. In this review, we attempted to address the involvement of FCSPs in the stimulation/downregulation of immune response cell signaling. The structural complexity and its foremost influential factor, extraction techniques, have also attracted attention, with concise details on the structural implications of bioactivity.

Protein O-fucosyltransferase 2 (PoFUT2) is an inverting glycosyltransferase (GT) that fucosylates thrombospondin repeats (TSRs) from group 1 and 2. PoFUT2 recognizes a large and diverse number of TSRs through a dynamic network of water-mediated interactions. By X-ray structural studies of C. elegans PoFUT2 complexed to a TSR of group 2, we demonstrate that this GT recognizes similarly the 3D structure of TSRs from both groups 1 and 2. Its active site is highly exposed to the solvent, suggesting that water molecules might also play an essential role in the fucosylation mechanism. We applied QM/MM methods using human PoFUT2 as a model, and found that HsPoFUT2 follows a classical S N 2 reaction mechanism in which water molecules contribute to a great extent in facilitating the release of the leaving pyrophosphate unit, causing the H transfer from the acceptor nucleophile (Thr/Ser) to the catalytic base, which is the last event in the reaction. This demonstrates the importance of water molecules not only in recognition of the ligands but also in catalysis., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

The biosynthesis of glycans is a highly conserved biological process and found in all domains of life. The expression of cell surface glycans is increasingly recognized as a target for therapeutic intervention given the role of glycans in major pathologies such as cancer and microbial infection. Herein, we summarize our contributions to the development of unnatural monosaccharide derivatives to infiltrate and alter the expression of both mammalian and bacterial glycans and their therapeutic application.

Peroxidative depolymerization is often used to elucidate the structure and structure-activity relationship of fucosylated glycosaminoglycan (FG), while the selectivity of bond cleavage and structural characteristics of the resulting fragments remain to be confirmed. Here, the FG from Stichopus variegatus (SvFG) was depolymerized by H 2 O 2 , and a series of yielded mono- and oligo-saccharides were purified. Almost all the non-reducing ends of oligosaccharides were d-GalNAc 4S6S , suggesting that GlcA-β1,3-GalNAc 4S6S linkage was preferentially cleaved. The model reactions showed the glycosidic bond of uronate was more susceptible than those of N-acetyl hexosamine and fucose, which should be due to bond energy of the anomeric CH. The reducing ends of oligosaccharides include C4-C6 saccharic acid and GalNAc or GalNAcA, which should be derived from the oxidation of the reducing end. A hexasaccharide with tartaric acid exhibited increased anti-iXase activity, suggesting the oxidation of reducing end did not impair the anti-iXase activity of FG-derived oligosaccharides., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Multidrug-resistant pathogens such as Burkholderia cenocepacia have become a hazard in the context of healthcare-associated infections, especially for patients admitted with cystic fibrosis or immuno-compromising conditions. Like other opportunistic Gram-negative bacteria, this pathogen establishes virulence and biofilms through lectin-mediated adhesion. In particular, the superlectin BC2L-C is believed to cross-link human epithelial cells to B. cenocepacia during pulmonary infections. We aimed to obtain glycomimetic antagonists able to inhibit the interaction between the N -terminal domain of BC2L-C (BC2L-C-Nt) and its target fucosylated human oligosaccharides. In a previous study, we identified by fragment virtual screening and validated a small set of molecular fragments that bind BC2L-C-Nt in the vicinity of the fucose binding site. Here, we report the rational design and synthesis of bifunctional C - or N -fucosides, generated by connecting these fragments to a fucoside core using a panel of rationally selected linkers. A modular route starting from two key fucoside intermediates was implemented for the synthesis, followed by evaluation of the new compounds as BC2L-C-Nt ligands with a range of techniques (surface plasmon resonance, isothermal titration calorimetry, saturation transfer difference NMR, differential scanning calorimetry, and X-ray crystallography). This study resulted in a hit molecule with an order of magnitude gain over the starting methyl fucoside and in two crystal structures of antagonist/lectin complexes.

Fucosylated chondroitin sulfate (FCS) from the sea cucumber Acaudina molpadioides (FCS Am ) is the first one that was reported to be branched by disaccharide GalNAc-(α1,2)-Fuc 3S4S (15%) and sulfated Fuc (85%). Here, four size-homogenous fractions, and seven oligosaccharides, were separated from its β-eliminative depolymerized products. Detailed NMR spectroscopic and MS analyses revealed the oligomers as hexa-, hepta-, octa-, and nonasaccharide, which further confirmed the precise structure of native FCS Am : it was composed of the CS-E-like backbone with a full content of sulfation at O-4 and O-6 of GalNAc in the disaccharide repeating unit, and the branches consisting of sulfated fucose (Fuc 4S and Fuc 2S4S ) and heterodisaccharide [GalNAc-(α1,2)-Fuc 3S4S ]. Pharmacologically, FCS Am and its depolymerized derivatives, including fractions and oligosaccharides, showed potent neurite outgrowth-promoting activity in a chain length-dependent manner. A comparison of analyses among oligosaccharides revealed that the sulfate pattern of the Fuc branches, instead of the heterodisaccharide, could affect the promotion intensity. Fuc 2S4S and the saccharide length endowed the neurite outgrowth stimulation activity most.