Analysis of endogenous NOTCH1 from POFUT1 S162L patient fibroblasts reveals the importance of the O-fucose modification on EGF12 in human development.

NOTCH1 (N1) is a transmembrane receptor interacting with membrane-tethered ligands on opposing cells that mediate the direct cell-cell interaction necessary for many cell fate decisions. Protein O-fucosyltransferase 1 (POFUT1) adds O-fucose to Epidermal Growth Factor (EGF)-like repeats in the NOTCH1 extracellular domain, which is required for trafficking and signaling activation. We previously showed that POFUT1 S162L caused a 90% loss of POFUT1 activity and global developmental defects in a patient; however, the mechanism by which POFUT1 contributes to these symptoms is still unclear. Compared to controls, POFUT1 S162L patient fibroblast cells had an equivalent amount of N1 on the cell surface but showed a 60% reduction of DLL1 ligand binding and a 70% reduction in JAG1 ligand binding. To determine if the reduction of O-fucose on N1 in POFUT1 S162L patient fibroblasts was the cause of these effects, we immunopurified endogenous N1 from control and patient fibroblasts and analyzed O-fucosylation using mass spectral glycoproteomics methods. N1 EGF8 to EGF12 comprise the ligand binding domain, and O-fucose on EGF8 and EGF12 physically interact with ligands to enhance affinity. Glycoproteomics of N1 from POFUT1 S162L patient fibroblasts showed WT fucosylation levels at all sites analyzed except for a large decrease at EGF9 and the complete absence of O-fucose at EGF12. Since the loss of O-fucose on EGF12 is known to have significant effects on N1 activity, this may explain the symptoms observed in the POFUT1 S162L patient.

Regulation of intracellular activity of N-glycan branching enzymes in mammals.

Most proteins in the secretory pathway are glycosylated, and N-glycans are estimated to be attached to over 7000 proteins in humans. As structural variation of N-glycans critically regulates the functions of a particular glycoprotein, it is pivotal to understand how structural diversity of N-glycans is generated in cells. One of the major factors conferring structural variation of N-glycans is the variable number of N-acetylglucosamine branches. These branch structures are biosynthesized by dedicated glycosyltransferases, including GnT-III (MGAT3), GnT-IVa (MGAT4A), GnT-IVb (MGAT4B), GnT-V (MGAT5), and GnT-IX (GnT-Vb, MGAT5B). In addition, the presence or absence of core modification of N-glycans, namely, core fucose (included as an N-glycan branch in this manuscript), synthesized by FUT8, also confers large structural variation on N-glycans, thereby crucially regulating many protein-protein interactions. Numerous biochemical and medical studies have revealed that these branch structures are involved in a wide range of physiological and pathological processes. However, the mechanisms regulating the activity of the biosynthetic glycosyltransferases are yet to be fully elucidated. In this review, we summarize the previous findings and recent updates regarding regulation of the activity of these N-glycan branching enzymes. We hope that such information will help readers to develop a comprehensive overview of the complex system regulating mammalian N-glycan maturation.

Hydroxysteroid 17-ß dehydrogenase 14 (HSD17B14) is an L-fucose dehydrogenase, the initial enzyme of the L-fucose degradation pathway.

L-Fucose (6-deoxy-L-galactose), a monosaccharide abundant in glycolipids and glycoproteins produced by mammalian cells, has been extensively studied for its role in intracellular biosynthesis and recycling of GDP-L-fucose for fucosylation. However, in certain mammalian species, L-fucose is efficiently broken down to pyruvate and lactate in a poorly understood metabolic pathway. In the 1970s, L-fucose dehydrogenase, an enzyme responsible for the initial step of this pathway, was partially purified from pig and rabbit livers and characterized biochemically. However, its molecular identity remained elusive until recently. This study reports the purification, identification, and biochemical characterization of the mammalian L-fucose dehydrogenase. The enzyme was purified from rabbit liver approximately 340-fold. Mass spectrometry analysis of the purified protein preparation identified mammalian hydroxysteroid 17-ß dehydrogenase 14 (HSD17B14) as the sole candidate enzyme. Rabbit and human HSD17B14 were expressed in HEK293T and Escherichia coli, respectively, purified and demonstrated to catalyze the oxidation of L-fucose to L-fucono-1,5-lactone, as confirmed by mass spectrometry and NMR analysis. Substrate specificity studies revealed that L-fucose is the preferred substrate for both enzymes. The human enzyme exhibited a catalytic efficiency for L-fucose that was 359-fold higher than its efficiency for estradiol. Additionally, recombinant rat HSD17B14 exhibited negligible activity towards L-fucose, consistent with the absence of L-fucose metabolism in this species. The identification of the gene encoding mammalian L-fucose dehydrogenase provides novel insights into the substrate specificity of enzymes belonging to the 17-ß-hydroxysteroid dehydrogenase family. This discovery also paves the way for unraveling the physiological functions of the L-fucose degradation pathway, which remains enigmatic.

Mutations in the SLC35C1 gene, contributing to significant differences in fucosylation patterns, may underlie the diverse phenotypic manifestations observed in leukocyte adhesion deficiency type II patients.

Congenital disorders of glycosylation (CDG) are a large family of genetic diseases resulting from defects in the synthesis of glycans and the attachment of glycans to macromolecules. The CDG known as leukocyte adhesion deficiency II (LAD II) is an autosomal, recessive disorder caused by mutations in the SLC35C1 gene, encoding a transmembrane protein of the Golgi apparatus, involved in GDP-fucose transport from the cytosol to the Golgi lumen. In this study, a cell-based model was used as a tool to characterize the molecular background of a therapy based on a fucose-supplemented diet. Such therapies have been successfully introduced in some (but not all) known cases of LAD II. In this study, the effect of external fucose was analyzed in SLC35C1 KO cell lines, expressing 11 mutated SLC35C1 proteins, previously discovered in patients with an LAD II diagnosis. For many of them, the cis-Golgi subcellular localization was affected; however, some proteins were localized properly. Additionally, although mutated SLC35C1 caused different α-1-6 core fucosylation of N-glycans, which explains previously described, more or less severe disorder symptoms, the differences practically disappeared after external fucose supplementation, with fucosylation restored to the level observed in healthy cells. This indicates that additional fucose in the diet should improve the condition of all patients. Thus, for patients diagnosed with LAD II we advocate careful analysis of particular mutations using the SLC35C1-KO cell line-based model, to predict changes in localization and fucosylation rate. We also recommend searching for additional mutations in the human genome of LAD II patients, when fucose supplementation does not influence patients' state.

Exploring the diverse biological significance and roles of fucosylated oligosaccharides.

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

Aberrant glycosylation of secretory mucin from the oral cavity in tobacco consumers: a pilot study.

Mucins are a family of high-molecular-weight O-linked glycoproteins which are the primary structural components of mucus and maintain homeostasis in the oral cavity. The present study was conducted as the first step towards establishing a correlation of aberrant mucin glycosylation with tobacco-associated clinical conditions. Tobacco habituates for the study were identified on the basis of type, duration, amount, and frequency of using tobacco products. The secretory mucin and its saccharides were determined from the saliva collected from smokers, smokeless tobacco habituates, and healthy, nonsmoking individuals. On the one hand, the salivary mucin content was markedly reduced in smokeless tobacco habituates with respect to smokers. On the other hand, the amount of sialic acid and fucose moieties of salivary mucin was increased in both smokers and smokeless tobacco habituates compared to the healthy cohort. Furthermore, the duration of tobacco exposure have been identified as the main factor influencing the extent of damage to the oral mucosa in terms of mucin secretion. The reduced secretory mucin content with aberrant glycosylation in the oral cavity may have a significant role in the further development or progression of oral diseases.

D-mannose as a new therapy for fucokinase deficiency-related congenital disorder of glycosylation (FCSK-CDG).

INTRODUCTION: Fucokinase deficiency-related congenital disorder of glycosylation (FCSK-CDG) is a rare autosomal recessive inborn error of metabolism characterized by a decreased flux through the salvage pathway of GDP-fucose biosynthesis due to a block in the recycling of L-fucose that exits the lysosome. FCSK-CDG has been described in 5 individuals to date in the medical literature, with a phenotype comprising global developmental delays/intellectual disability, hypotonia, abnormal myelination, posterior ocular disease, growth and feeding failure, immune deficiency, and chronic diarrhea, without clear therapeutic recommendations. PATIENT AND METHODS: In a so far unreported FCSK-CDG patient, we studied proteomics and glycoproteomics in vitro in patient-derived fibroblasts and also performed in vivo glycomics, before and after treatment with either D-Mannose or L-Fucose. RESULTS: We observed a marked increase in fucosylation after D-mannose supplementation in fibroblasts compared to treatment with L-Fucose. The patient was then treated with D-mannose at 850 mg/kg/d, with resolution of the chronic diarrhea, resolution of oral aversion, improved weight gain, and observed developmental gains. Serum N-glycan profiles showed an improvement in the abundance of fucosylated glycans after treatment. No treatment-attributed adverse effects were observed. CONCLUSION: D-mannose is a promising new treatment for FCSK-CDG.

A Computational and Chemical Design Strategy for Manipulating Glycan-Protein Recognition.

Glycans are complex biomolecules that encode rich information and regulate various biological processes, such as fertilization, host-pathogen binding, and immune recognition, through interactions with glycan-binding proteins. A key driving force for glycan-protein recognition is the interaction between the π electron density of aromatic amino acid side chains and polarized C─H groups of the pyranose (termed the CH-π interaction). However, the relatively weak binding affinity between glycans and proteins has hindered the application of glycan detection and imaging. Here, computational modeling and molecular dynamics simulations are employed to design a chemical strategy that enhances the CH-π interaction between glycans and proteins by genetically incorporating electron-rich tryptophan derivatives into a lectin PhoSL, which specifically recognizes core fucosylated N-linked glycans. This significantly enhances the binding affinity of PhoSL with the core fucose ligand and enables sensitive detection and imaging of core fucosylated glycans in vitro and in xenograft tumors in mice. Further, the study showed that this strategy is applicable to improve the binding affinity of GafD lectin for N-acetylglucosamine-containing glycans. The approach thus provides a general and effective way to manipulate glycan-protein recognition for glycoscience applications.

L-fucose and fucoidan alleviate high-salt diet-promoted acute inflammation.

Excessive salt intake is a widespread health issue observed in almost every country around the world. A high salt diet (HSD) has a strong correlation with numerous diseases, including hypertension, chronic kidney disease, and autoimmune disorders. However, the mechanisms underlying HSD-promotion of inflammation and exacerbation of these diseases are not fully understood. In this study, we observed that HSD consumption reduced the abundance of the gut microbial metabolite L-fucose, leading to a more substantial inflammatory response in mice. A HSD led to increased peritonitis incidence in mice, as evidenced by the increased accumulation of inflammatory cells and elevated levels of inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and monocyte chemotactic protein-1 (MCP-1, also known as C-C motif chemokine ligand 2 or CCL2), in peritoneal lavage fluid. Following the administration of broad-spectrum antibiotics, HSD-induced inflammation was abolished, indicating that the proinflammatory effects of HSD were not due to the direct effect of sodium, but rather to HSD-induced alterations in the composition of the gut microbiota. By using untargeted metabolomics techniques, we determined that the levels of the gut microbial metabolite L-fucose were reduced by a HSD. Moreover, the administration of L-fucose or fucoidan, a compound derived from brown that is rich in L-fucose, normalized the level of inflammation in mice following HSD induction. In addition, both L-fucose and fucoidan inhibited LPS-induced macrophage activation in vitro. In summary, our research showed that reduced L-fucose levels in the gut contributed to HSD-exacerbated acute inflammation in mice; these results indicate that L-fucose and fucoidan could interfere with HSD-promotion of the inflammatory response.

Molecular insights into FucR transcription factor to control the metabolism of L-fucose in Bifidobacterium longum subsp. infantis.

Bifidobacterium longum subsp. infantis commonly colonizes the human gut and is capable of metabolizing L-fucose, which is abundant in the gut. Multiple studies have focused on the mechanisms of L-fucose utilization by B. longum subsp. infantis, but the regulatory pathways governing the expression of these catabolic processes are still unclear. In this study, we have conducted a structural and functional analysis of L-fucose metabolism transcription factor FucR derived from B. longum subsp. infantis Bi-26. Our results indicated that FucR is a L-fucose-sensitive repressor with more α-helices, fewer ß-sheets, and ß-turns. Transcriptional analysis revealed that FucR displays weak negative self-regulation, which is counteracted in the presence of L-fucose. Isothermal titration calorimetry indicated that FucR has a 2:1 stoichiometry with L-fucose. The key amino acid residues for FucR binding L-fucose are Asp280 and Arg331, with mutation of Asp280 to Ala resulting in a decrease in the affinity between FucR and L-fucose with the Kd value from 2.58 to 11.68 µM, and mutation of Arg331 to Ala abolishes the binding ability of FucR towards L-fucose. FucR specifically recognized and bound to a 20-bp incomplete palindrome sequence (5'-ACCCCAATTACGAAAATTTTT-3'), and the affinity of the L-fucose-loaded FucR for the DNA fragment was lower than apo-FucR. The results provided new insights into the regulating L-fucose metabolism by B. longum subsp. infantis.

The functional role of L-fucose on dendritic cell function and polarization.

Despite significant advances in the development and refinement of immunotherapies administered to combat cancer over the past decades, a number of barriers continue to limit their efficacy. One significant clinical barrier is the inability to mount initial immune responses towards the tumor. As dendritic cells are central initiators of immune responses in the body, the elucidation of mechanisms that can be therapeutically leveraged to enhance their functions to drive anti-tumor immune responses is urgently needed. Here, we report that the dietary sugar L-fucose can be used to enhance the immunostimulatory activity of dendritic cells (DCs). L-fucose polarizes immature myeloid cells towards specific DC subsets, specifically cDC1 and moDC subsets. In vitro, L-fucose treatment enhances antigen uptake and processing of DCs. Furthermore, our data suggests that L-fucose-treated DCs increase stimulation of T cell populations. Consistent with our functional assays, single-cell RNA sequencing of intratumoral DCs from melanoma- and breast tumor-bearing mice confirmed transcriptional regulation and antigen processing as pathways that are significantly altered by dietary L-fucose. Together, this study provides the first evidence of the ability of L-fucose to bolster DC functionality and provides rational to further investigate how L-fucose can be used to leverage DC function in order to enhance current immunotherapy.

Up regulation of serum L fucose glycoprotein as a diagnostic biomarker for dysplasia in oral sub mucous fibrosis patients.

Glycoproteins, essential for cellular functions, contain monosaccharides like Levo-fucose, crucial for cell communication. Recent research highlights serum L-fucose as a potential biomarker for early detection of malignancies. Typically, serum L-fucose levels are low but rise with malignancy. This study evaluates serum L-fucose as an early biomarker in oral submucous fibrosis (OSMF) patients. Aim: Assess serum L-fucose's diagnostic potential for dysplasia in OSMF patients. Objectives: Determine the Association between Serum L Fucose Glycoprotein Levels and Dysplasia in OSF Patients.Evaluate the Diagnostic Accuracy of Serum L Fucose Glycoprotein as a Biomarker for OSF-Related Dysplasia. Methodology: Over a span of two years, this study encompassed 80 subjects, aged between 18 and 60 years, who were clinically and histopathologically identified as OSMF patients, with or without dysplastic alterations. From each participant, 5 ml of blood was collected. Following centrifugation to separate the serum, the samples were analyzed to determine the levels of Levo-fucose. Statistical analysis: Using SPSS (version 17.0), serum L-Fucose levels of the case group were compared to the control group using ANOVA. Frequencies were analyzed with the chi-square test, and Tukey's Test was used for multiple comparisons. Significance was set at p < 0.01. Results: The analysis revealed a statistically significant disparity in the mean serum L-Fucose levels between the two groups (p < 0.01). Notably, Group II patients (those with OSMF and dysplasia) exhibited markedly elevated mean serum L-fucose levels. Conclusion: Elevated serum L-Fucose levels were observed in OSMF patients with dysplasia. Harmful habits, especially gutkha chewing, were linked to Oral Squamous Cell Carcinoma onset. Serum L-fucose can be a reliable marker for evaluating precancerous conditions.

Exploiting the biocatalytic potential of co-expressed l-fucose isomerase and d-tagatose 3-epimerase for the biosynthesis of 6-deoxy-l-sorbose.

6-Deoxy-l-sorbose (6-DLS) is an imperative rare sugar employed in food, agriculture, pharmaceutical and cosmetic industeries. However, it is a synthetic and very expensive rare sugars, previously synthesized by chemo-enzymatic methods through a long chain of chemical processes. Recently, enzymatic synthesis of rare sugars has attracted a lot of attention due to its advantages over synthetic methods. In this work, a promising approach for the synthesis of 6-DLS from an inexpensive sugar l-fucose was identified. The genes for l-fucose isomerase from Paenibacillus rhizosphaerae (Pr-LFI) and genes for d-tagatose-3-epimerase from Caballeronia fortuita (Cf-DTE) have been used for cloning and co-expression in Escherichia coli, developed a recombinant plasmid harboring pANY1-Pr-LFI/Cf-DTE vector. The recombinant co-expression system exhibited an optimum activity at 50 °C of temperature and pH 6.5 in the presence of Co2+ metal ion which inflated the catalytic activity by 6.8 folds as compared to control group with no metal ions. The recombinant co-expressed system was stable up to more than 50 % relative activity after 12 h and revealed a melting temperature (Tm) of 63.38 °C exhibiting half-life of 13.17 h at 50 °C. The co-expression system exhibited, 4.93, 11.41 and 16.21 g/L of 6-DLS production from initial l-fucose concentration of 30, 70 and 100 g/L, which equates to conversion yield of 16.44 %, 16.30 % and 16.21 % respectively. Generally, this study offers a promising strategy for the biological production of 6-DLS from an inexpensive substrate l-fucose in slightly acidic conditions with the aid of co-expression system harboring Pr-LFI and CF-DTE genes.

Efficient Biosynthesis of Difucosyllactose via De Novo GDP-l-Fucose Pathway in Metabolically Engineered Escherichia coli.

Difucosyllactose (DFL) is an important component of human milk oligosaccharides (HMOs) and has significant benefits for the growth and development of infants. So far, a few microbial cell factories have been constructed for the production of DFL, which still have problems of low production and high cost. Herein, a high-level de novo pathway DFL-producing strain was constructed by multistep optimization strategies in Escherichia coli BL21star(DE3). We first efficiently synthesized the intermediate 2'-fucosyllactose (2'-FL) in E. coli BL21star(DE3) by the advisable stepwise strategy. The truncated α-1,3/4-fucosyltransferase (Hp3/4FT) was then introduced into the engineered strain to achieve de novo biosynthesis of DFL. ATP-dependent protease (Lon) and GDP-mannose hydrolase (NudK) were deleted, and mannose-6-phosphate isomerase (ManA) was overexpressed to improve GDP-l-fucose accumulation. The regulator RcsA was overexpressed to fine-tune the expression level of pathway genes, thereby increasing the synthesis of DFL. The final strain produced 6.19 g/L of DFL in the shake flask and 33.45 g/L of DFL in the 5 L fermenter, which were the highest reported titers so far. This study provides a more economical, sustainable, and effective strategy to produce the fucosylated human milk oligosaccharides (HMOs).

Structure, Physicochemical Properties and Biological Activity of Lipopolysaccharide from the Rhizospheric Bacterium Ochrobactrum quorumnocens T1Kr02, Containing d-Fucose Residues.

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)-ß-d-Fucf-(1→3)-ß-d-Fucp-(1→. The structure of the periplasmic glucan coextracted with LPS was established by NMR spectroscopy and chemical methods: →2)-ß-d-Glcp-(1→. Non-stoichiometric modifications were identified in both polysaccharides: 50% of d-fucofuranose residues at position 3 were O-acetylated, and 15% of d-Glcp 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.

A Fucose-Containing Sulfated Polysaccharide from Spatoglossum schröederi Potentially Targets Tumor Growth Rather Than Cytotoxicity: Distinguishing Action on Human Melanoma Cell Lines.

Natural substances are strategic candidates for drug development in cancer research. Marine-derived molecules are of special interest due to their wide range of biological activities and sustainable large-scale production. Melanoma is a type of skin cancer that originates from genetic mutations in melanocytes. BRAF, RAS, and NF1 mutations are described as the major melanoma drivers, but approximately 20% of patients lack these mutations and are included in the triple wild-type (tripleWT) classification. Recent advances in targeted therapy directed at driver mutations along with immunotherapy have only partially improved patients' overall survival, and consequently, melanoma remains deadly when in advanced stages. Fucose-containing sulfated polysaccharides (FCSP) are potential candidates to treat melanoma; therefore, we investigated Fucan A, a FCSP from Spatoglossum schröederi brown seaweed, in vitro in human melanoma cell lines presenting different mutations. Up to 72 h Fucan A treatment was not cytotoxic either to normal melanocytes or melanoma cell lines. Interestingly, it was able to impair the tripleWT CHL-1 cell proliferation (57%), comparable to the chemotherapeutic cytotoxic drug cisplatin results, with the advantage of not causing cytotoxicity. Fucan A increased CHL-1 doubling time, an effect attributed to cell cycle arrest. Vascular mimicry, a close related angiogenesis process, was also impaired (73%). Fucan A mode of action could be related to gene expression modulation, in special ß-catenin downregulation, a molecule with protagonist roles in important signaling pathways. Taken together, results indicate that Fucan A is a potential anticancer molecule and, therefore, deserves further investigation.

Elevated fucose content enhances the cryoprotective performance of anionic polysaccharides.

Biological cryopreservation often involves using a cryoprotective agent (CPA) to mitigate lethal physical stressors cells endure during freezing and thawing, but effective CPA concentrations are cytotoxic. Hence, natural polysaccharides have been studied as biocompatible alternatives. Here, a subset of 26 natural polysaccharides of various chemical composition was probed for their potential in enhancing the metabolic post-thaw viability (PTV) of cryopreserved Vero cells. The best performing cryoprotective polysaccharides contained significant fucose amounts, resulting in average PTV 2.8-fold (up to 3.1-fold) compared to 0.8-fold and 2.2-fold for all non-cryoprotective and cryoprotective polysaccharides, respectively, outperforming the optimized commercial CryoStor™ CS5 formulation (2.6-fold). Stoichiometrically, a balance between fucose (18-35.7 mol%), uronic acids (UA) (13.5-26 mol%) and high molecular weight (MW > 1 MDa) generated optimal PTV. Principal component analysis (PCA) revealed that fucose enhances cell survival by a charge-independent, MW-scaling mechanism (PC1), drastically different from the charge-dominated ice growth disruption of UA (PC2). Its neutral nature and unique properties distinguishable from other neutral monomers suggest fucose may play a passive role in conformational adaptability of polysaccharide to ice growth inhibition, or an active role in cell membrane stabilization through binding. Ultimately, fucose-rich anionic polysaccharides may indulge in polymer-ice and polymer-cell interactions that actively disrupt ice and minimize lethal volumetric fluctuations due to a balanced hydrophobic-hydrophilic character. Our research showed the critical role neutral fucose plays in enhancing cellular cryopreservation outcomes, disputing previous assumptions of polyanionicity being the sole governing predictor of cryoprotection.

α-L-Fucosidases from an Alpaca Faeces Metagenome: Characterisation of Hydrolytic and Transfucosylation Potential.

In various life forms, fucose-containing glycans play vital roles in immune recognition, developmental processes, plant immunity, and host-microbe interactions. Together with glucose, galactose, N-acetylglucosamine, and sialic acid, fucose is a significant component of human milk oligosaccharides (HMOs). Fucosylated HMOs benefit infants by acting as prebiotics, preventing pathogen attachment, and potentially protecting against infections, including HIV. Although the need for fucosylated derivatives is clear, their availability is limited. Therefore, synthesis methods for various fucosylated oligosaccharides are explored, employing enzymatic approaches and α-L-fucosidases. This work aimed to characterise α-L-fucosidases identified in an alpaca faeces metagenome. Based on bioinformatic analyses, they were confirmed as members of the GH29A subfamily. The recombinant α-L-fucosidases were expressed in Escherichia coli and showed hydrolytic activity towards p-nitrophenyl-α-L-fucopyranoside and 2'-fucosyllactose. Furthermore, the enzymes' biochemical properties and kinetic characteristics were also determined. All four α-L-fucosidases could catalyse transfucosylation using a broad diversity of fucosyl acceptor substrates, including lactose, maltotriose, L-serine, and L-threonine. The results contribute insights into the potential use of α-L-fucosidases for synthesising fucosylated amino acids.

Distinctive domains and activity regulation of core fucosylation enzyme FUT8.

BACKGROUND: Core fucose, a structure added to the reducing end N-acetylglucosamine of N-glycans, has been shown to regulate various physiological and pathological processes, including melanoma metastasis, exacerbation of chronic obstructive pulmonary disease, and severe outcomes in COVID-19. SCOPE OF REVIEW: Recent research has shed light on regulation of the activity and subcellular localization of a1,6-fucosyltransferase (FUT8), the glycosyltransferase responsible for core fucose biosynthesis, unraveling the mechanisms for controlling core fucosylation in vivo. MAJOR CONCLUSIONS: This review summarizes the various features of FUT8, including its domains, structures, and substrate specificity. Additionally, we discuss the potential involvement of FUT8-binding proteins, such as oligosaccharyltransferase subunits, in the regulation of FUT8 activity, substrate specificity, and the secretion of FUT8. GENERAL SIGNIFICANCE: We anticipate that this review will contribute to a deeper understanding of the control of core fucose levels in vivo and involvement of core fucosylation in FUT8-relevant functions and diseases.

Analysis of the Healthy Platelet Proteome Identifies a New Form of Domain-Specific O-Fucosylation.

Platelet activation induces the secretion of proteins that promote platelet aggregation and inflammation. However, detailed analysis of the released platelet proteome is hampered by platelets' tendency to preactivate during their isolation and a lack of sensitive protocols for low abundance releasate analysis. Here, we detail the most sensitive analysis to date of the platelet releasate proteome with the detection of >1300 proteins. Unbiased scanning for posttranslational modifications within releasate proteins highlighted O-glycosylation as being a major component. For the first time, we detected O-fucosylation on previously uncharacterized sites including multimerin-1 (MMRN1), a major alpha granule protein that supports platelet adhesion to collagen and is a carrier for platelet factor V. The N-terminal elastin microfibril interface (EMI) domain of MMRN1, a key site for protein-protein interaction, was O-fucosylated at a conserved threonine within a new domain context. Our data suggest that either protein O-fucosyltransferase 1, or a novel protein O-fucosyltransferase, may be responsible for this modification. Mutating this O-fucose site on the EMI domain led to a >50% reduction of MMRN1 secretion, supporting a key role of EMI O-fucosylation in MMRN1 secretion. By comparing releasates from resting and thrombin-treated platelets, 202 proteins were found to be significantly released after high-dose thrombin stimulation. Complementary quantification of the platelet lysates identified >3800 proteins, which confirmed the platelet origin of releasate proteins by anticorrelation analysis. Low-dose thrombin treatment yielded a smaller subset of significantly regulated proteins with fewer secretory pathway enzymes. The extensive platelet proteome resource provided here (larancelab.com/platelet-proteome) allows identification of novel regulatory mechanisms for drug targeting to address platelet dysfunction and thrombosis.