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[Display omitted] • Lipidomics identified 23 lipid classes low in glycolipids in tick salivary glands. • Phosphatidylcholine (PC; 26.66%) was the lipid class with the highest representation. • Tick salivary glycolipids were mostly represented by diacylglycerol (DG; 0.48%). • Individuals bitten by ticks produced more phosphatidylcholine-IgG antibodies. • DG levels were higher in tick salivary glands than in tick saliva. Ticks, as hematophagous ectoparasites, can manipulate host immune and metabolic processes, causing tick-borne allergies such as α-Gal syndrome (AGS). Glycolipids with bound galactose-alpha-1–3-galactose (α-Gal) are potential allergenic molecules associated with AGS. Nevertheless, proteins and lipids lacking α-Gal modifications may contribute to tick salivary allergies and be linked to AGS. In this study, we characterized the effect of deglycosylated tick salivary proteins without lipids on treated zebrafish fed with dog food formulated with mammalian (beef, lamb, pork) meat by quantitative proteomics analysis of intestinal samples. The characterization and functional annotations of tick salivary lipids with low representation of glycolipids was conducted using a lipidomics approach. Results showed a significant effect of treatment with saliva and saliva deglycosylated protein fraction on zebrafish abnormal or no feeding (p < 0.005). Treatment with this fraction affected multiple metabolic pathways, defense responses to pathogens and protein metabolism, which correlated with abnormal or no feeding. Lipidomics analysis identified 23 lipid classes with low representation of glycolipids (0.70% of identified lipids). The lipid class with highest representation was phosphatidylcholine (PC; 26.66%) and for glycolipids it corresponded to diacylglycerol (DG; 0.48%). Qualitative analysis of PC antibodies revealed that individuals bitten by ticks were more likely to produce PC-IgG antibodies (p < 0.001). DG levels were significantly higher in tick salivary glands (p < 0.05) compared with tick saliva and salivary fractions. The α-Gal content was higher in tick saliva than in deglycosylated saliva and lipid fractions. These results support a possible role for tick salivary proteins and lipids without α-Gal modifications in AGS. [ABSTRACT FROM AUTHOR]

Breast milk is a vital source of nutrients, prebiotics, probiotics, and protective factors, including antibodies, immune cells and antimicrobial proteins. Using bacterial lipopolysaccharide arrays, we investigated the reactivity and specificity of breast milk antibodies towards microbial antigens, comparing samples from rural Kenya and urban Switzerland. Results showed considerable variability in antibody reactivity both within and between these locations. Kenyan breast milk demonstrated broad reactivity to bacterial lipopolysaccharides, likely due to increased microbial exposure. Antibodies primarily recognized the O-antigens of lipopolysaccharides and showed strong binding to specific carbohydrate motifs. Notably, antibodies against specific Escherichia coli O-antigens showed cross-reactivity with parasitic pathogens like Leishmania major and Plasmodium falciparum, thus showing that antibodies reacting against lipopolysaccharide Oantigens can recognize a wide range of antigens beyond bacteria. The observed diversity in antigen recognition highlights the significance of breast milk in safeguarding infants from infections, particularly those prevalent in specific geographic regions. The findings also offer insights for potential immunobiotic strategies to augment natural antibody-mediated defense against diverse pathogens. [ABSTRACT FROM AUTHOR]

Effective use of adeno-associated viruses (AAVs) for clinical gene therapy is limited by their propensity to accumulate in and transduce the liver. This natural liver tropism is associated with severe adverse events at the high doses that can be necessary for achieving therapeutic transgene expression in extrahepatic tissues. To improve the safety and cost of AAV gene therapy, capsid engineering efforts are underway to redirect in vivo AAV biodistribution away from the liver toward disease-relevant peripheral organs such as the heart. Building on previous work, we generated a series of AAV libraries containing variations at three residues (Y446, N470, and W503) of the galactose-binding pocket of the AAV9 VP1 protein. Screening of this library in mice identified the XRH family of variants (Y446X, N470R, and W503H), the strongest of which, HRH, exhibited a 6-fold reduction in liver RNA expression and a 10-fold increase in cardiac RNA expression compared with wild-type AAV9 in the mouse. Screening of our library in a nonhuman primate (NHP) revealed reduced performance of AAV9 and two closely related vectors in the NHP liver compared with the mouse liver. Measurement of the galactose-binding capacity of our library further identified those same three vectors as the only strong galactose binders, suggesting an altered galactose presentation between the mouse and NHP liver. N-glycan profiling of these tissues revealed a 9% decrease in exposed galactose in the NHP liver compared with the mouse liver. In this work, we identified a novel family of AAV variants with desirable biodistribution properties that may be suitable for targeting extrahepatic tissues such as the heart. These data also provide important insights regarding species- and tissue-specific differences in glycan presentation that may have implications for the development and translation of AAV gene therapies. [ABSTRACT FROM AUTHOR]

Cell surface components, specifically glycans, play a significant role in several biological functions like cell structure, crosstalk between cells, and eventual target recognition of the cells for therapeutics. The dense layer of glycans, i. e., glycocalyx, could differ in taxon, species, and cell type. Glycans are coupled with lipids and proteins to form glycolipids, glycoproteins, proteoglycans, and glycosylphosphatidylinositol‐anchored proteins, making their study challenging. However, understanding glycosylation at the cellular level is vital for fundamental research and advancing glycan‐targeted therapy. Among different pathways, metabolic glycan labelling uses the natural metabolic processes of the cell to introduce abiotic functionality into glycan residues. The Bertozzi group pioneered metabolic oligosaccharide engineering using glycan salvage pathways to convert monosaccharides with unnatural modifications. This eventually results in the probe becoming part of the complex cellular glycan structures via click chemistry using copper. On the other hand, the boronic acid‐based probe can recognise carbohydrates in a single step without any chemical modification of the surface. This review discusses the significance of glycans as biomarkers for different diseases and the necessity to evaluate them in situ within the physiological environment. The review also discusses the prospect of this field and its potential applications. [ABSTRACT FROM AUTHOR]

Glycans, carbohydrates, and glycoconjugates are involved in many crucial biological processes, such as disease development, immune responses, and cell–cell recognition. Glycans and carbohydrates are known for the large number of isomeric features associated with their structures, making analysis challenging compared with other biomolecules. Mass spectrometry has become the primary method of structural characterization for carbohydrates, glycans, and glycoconjugates. Metal adduction is especially important for the mass spectrometric analysis of carbohydrates and glycans. Metal‐ion adduction to carbohydrates and glycoconjugates affects ion formation and the three‐dimensional, gas‐phase structures. Herein, we discuss how metal‐ion adduction impacts ionization, ion mobility, ion activation and dissociation, and hydrogen/deuterium exchange for carbohydrates and glycoconjugates. We also compare the use of different metals for these various techniques and highlight the value in using metals as charge carriers for these analyses. Finally, we provide recommendations for selecting a metal for analysis of carbohydrate adducts and describe areas for continued research. [ABSTRACT FROM AUTHOR]

Sialyl‐Lewisx (SLex) is involved in immune regulation, human fertilization, cancer, and bacterial and viral diseases. The influence of the complex glycan structures, which can present SLex epitopes, on binding is largely unknown. We report here a chemoenzymatic strategy for the preparation of a panel of twenty‐two isomeric asymmetrical tri‐antennary N‐glycans presenting SLex‐Lex epitopes on either the MGAT4 or MGAT5 arm that include putative high‐affinity ligands for E‐selectin. The N‐glycans were prepared starting from a sialoglycopeptide isolated from egg yolk powder and took advantage of inherent substrate preferences of glycosyltransferases and the use of 5′‐diphospho‐N‐trifluoracetylglucosamine (UDP‐GlcNHTFA) that can be transferred by branching N‐acetylglucosaminyltransferases to give, after base treatment, GlcNH2‐containing glycans that temporarily disable an antenna from enzymatic modification. Glycan microarray binding studies showed that E‐selectin bound equally well to linear glycans and tri‐antennary N‐glycans presenting SLex‐Lex. On the other hand, it was found that hemagglutinins (HA) of H5 influenza A viruses (IAV) preferentially bound the tri‐antennary N‐glycans. Furthermore, several H5 HAs preferentially bound to N‐glycan presenting SLex on the MGAT4 arm. SLex is displayed in the respiratory tract of several avian species, demonstrating the relevance of investigating the binding of, among others IAVs, to complex N‐glycans presenting SLex. [ABSTRACT FROM AUTHOR]

Numerous viruses have been found to exploit glycoconjugates expressed on human cells as their initial attachment factor for viral entry and infection. The virus-cell glycointeractome, when characterized, may serve as a template for antiviral drug design. Heparan sulfate proteoglycans extensively decorate the human cell surface and were previously described as a primary receptor for human metapneumovirus (HMPV). After respiratory syncytial virus, HMPV is the second most prevalent respiratory pathogen causing respiratory tract infection in young children. To date, there is neither vaccine nor drug available to prevent or treat HMPV infection. Using a multidisciplinary approach, we report for the first time the glycointeractome of the HMPV fusion (F) protein, a viral surface glycoprotein that is essential for target-cell recognition, attachment, and entry. Our glycan microarray and surface plasmon resonance results suggest that Galβ1-3/4GlcNAc moieties that may be sialylated or fucosylated are readily recognized by HMPV F. The bound motifs are highly similar to the N-linked and O-linked glycans primarily expressed on the human lung epithelium. We demonstrate that the identified glycans have the potential to compete with the cellular receptors used for HMPV entry and consequently block HMPV infection. We found that lacto-N-neotetraose demonstrated the strongest HMPV binding inhibition in a cell infection assay. Our current findings offer an encouraging and novel avenue for the design of anti-HMPV drug candidates using oligosaccharide templates. IMPORTANCE All cells are decorated with a dense coat of sugars that makes a sugar code. Many respiratory viruses exploit this sugar code by binding to these sugars to cause infection. Human metapneumovirus is a leading cause for acute respiratory tract infections. Despite its medical importance, there is no vaccine or antiviral drug available to prevent or treat human metapneumovirus infection. This study investigates how human metapneumovirus binds to sugars in order to more efficiently infect the human host. We found that human metapneumovirus binds to a diverse range of sugars and demonstrated that these sugars can ultimately block viral infection. Understanding how viruses can take advantage of the sugar code on our cells could identify new intervention and treatment strategies to combat viral disease. [ABSTRACT FROM AUTHOR]

Most of Earth’s biomass is composed of polysaccharides. During biomass decomposition, polysaccharides are degraded by heterotrophic bacteria as a nutrient and energy source and are thereby partly remineralized into CO2. As polysaccharides are heterogeneously distributed in nature, following the colonization and degradation of a polysaccharide hotspot the cells need to reach new polysaccharide hotspots. Even though many studies indicate that these degradation-dispersal cycles contribute to the carbon flow in marine systems, we know little about how cells alternate between polysaccharide degradation and motility, and which environmental factors trigger this behavioral switch. Here, we studied the growth of the marine bacterium Vibrio cyclitrophicus ZF270 on the abundant marine polysaccharide alginate, both in its soluble polymeric form as well as on its breakdown products. We used microfluidics coupled to time-lapse microscopy to analyze motility and growth of individual cells, and RNA sequencing to study associated changes in gene expression. We found that single cells grow at reduced rate on alginate until they form large groups that cooperatively break down the polymer. Exposing cell groups to digested alginate accelerates cell growth and changes the expression of genes involved in alginate degradation and catabolism, central metabolism, ribosomal biosynthesis, and transport. However, exposure to digested alginate also triggers cells to become motile and disperse from cell groups, proportionally increasing with the group size before the nutrient switch, and this is accompanied by high expression of genes involved in flagellar assembly, chemotaxis, and quorum sensing. The motile cells chemotax toward polymeric but not digested alginate, likely enabling them to find new polysaccharide hotspots. Overall, our findings reveal cellular mechanisms that might also underlie bacterial degradation-dispersal cycles, which influence the remineralization of biomass in marine environments.

Summary: Staphylococcus aureus is a major human pathogen, yet the immune factors that protect against infection remain elusive. High titers of opsonic IgG antibodies, achieved in preclinical animal immunization studies, have consistently failed to provide protection in humans. Here, we investigate antibody responses to the conserved S. aureus surface glycan wall teichoic acid (WTA) and detect the presence of WTA-specific IgM and IgG antibodies in the plasma of healthy individuals. Functionally, WTA-specific IgM outperforms IgG in opsonophagocytic killing of S. aureus and protects against disseminated S. aureus bacteremia through passive immunization. In a clinical setting, patients with S. aureus bacteremia have significantly lower WTA-specific IgM but similar IgG levels compared to healthy controls. Importantly, low WTA-IgM levels correlate with disease mortality and impaired bacterial opsonization. Our findings may guide risk stratification of hospitalized patients and inform future design of antibody-based therapies and vaccines against serious S. aureus infection.

Breast milk is a vital source of nutrients, prebiotics, probiotics, and protective factors, including antibodies, immune cells and antimicrobial proteins. Using bacterial lipopolysaccharide arrays, we investigated the reactivity and specificity of breast milk antibodies towards microbial antigens, comparing samples from rural Kenya and urban Switzerland. Results showed considerable variability in antibody reactivity both within and between these locations. Kenyan breast milk demonstrated broad reactivity to bacterial lipopolysaccharides, likely due to increased microbial exposure. Antibodies primarily recognized the O-antigens of lipopolysaccharides and showed strong binding to specific carbohydrate motifs. Notably, antibodies against specific Escherichia coli O-antigens showed cross-reactivity with parasitic pathogens like Leishmania major and Plasmodium falciparum, thus showing that antibodies reacting against lipopolysaccharide O-antigens can recognize a wide range of antigens beyond bacteria. The observed diversity in antigen recognition highlights the significance of breast milk in safeguarding infants from infections, particularly those prevalent in specific geographic regions. The findings also offer insights for potential immunobiotic strategies to augment natural antibody-mediated defense against diverse pathogens.

BackgroundCurrent assessment of dietary carbohydrates does not adequately reflect the nutritional properties and effects on gut microbial structure and function. Deeper characterization of food carbohydrate composition can serve to strengthen the link between diet and gastrointestinal health outcomes.ObjectivesThe present study aims to characterize the monosaccharide composition of diets in a healthy US adult cohort and use these features to assess the relationship between monosaccharide intake, diet quality, characteristics of the gut microbiota, and gastrointestinal inflammation.MethodsThis observational, cross-sectional study enrolled males and females across age (18-33 y, 34-49 y, and 50-65 y) and body mass index (normal, 18.5-24.99 kg/m2; overweight, 25-29.99 kg/m2; and obese, 30-44 kg/m2) categories. Recent dietary intake was assessed by the automated self-administered 24-h dietary recall system, and gut microbiota were assessed with shotgun metagenome sequencing. Dietary recalls were mapped to the Davis Food Glycopedia to estimate monosaccharide intake. Participants with >75% of carbohydrate intake mappable to the glycopedia were included (N = 180).ResultsDiversity of monosaccharide intake was positively associated with the total Healthy Eating Index score (Pearson's r = 0.520, P = 1.2 × 10-13) and negatively associated with fecal neopterin (Pearson's r = -0.247, P = 3.0 × 10-3). Comparing high with low intake of specific monosaccharides revealed differentially abundant taxa (Wald test, P < 0.05), which was associated with the functional capacity to break down these monomers (Wilcoxon rank-sum test, P < 0.05).ConclusionsMonosaccharide intake was associated with diet quality, gut microbial diversity, microbial metabolism, and gastrointestinal inflammation in healthy adults. As specific food sources were rich in particular monosaccharides, it may be possible in the future to tailor diets to fine-tune the gut microbiota and gastrointestinal function. This trial is registered at www.Clinicaltrialsgov as NCT02367287.

Carrying the apolipoprotein E (ApoE) Ɛ4 allele is associated with an increased risk of cerebral amyloidosis and late-onset Alzheimer's disease, but the degree to which apoE glycosylation affects its development is not clear. In a previous pilot study, we identified distinct total and secondary isoform-specific cerebral spinal fluid (CSF) apoE glycosylation profiles, with the E4 isoform having the lowest glycosylation percentage (E2 > E3 > E4). In this work, we extend the analysis to a larger cohort of individuals (n = 106), utilizing matched plasma and CSF samples with clinical measures of AD biomarkers. The results confirm the isoform-specific glycosylation of apoE in CSF, resulting from secondary CSF apoE glycosylation patterns. CSF apoE glycosylation percentages positively correlated with CSF Aβ42 levels (r = 0.53, p  E3 and increased binding affinity to heparin. These results indicate that apoE glycosylation has a new and important role in influencing brain Aβ metabolism and can be a potential target of treatment.

Several studies have examined the impact of prebiotics on gut microbiota and associated changes in host physiology. Here, we used the in vitro cultivation of human fecal samples stimulated with a series of chemically related prebiotics and medicinal herbs commonly used in Ayurvedic medicine, followed by 16S rRNA sequencing. We applied a genome-wide metabolic reconstruction of enumerated communities to compare and contrast the structural and functional impact of prebiotics and medicinal herbs. In doings so, we examined the relationships between discrete variations in sugar composition and sugar linkages associated with each prebiotic to drive changes in microbiota composition. The restructuring of microbial communities with glycan substrates alters community metabolism and its potential impact on host physiology. We analyzed sugar fermentation pathways and products predicted to be formed and prebiotic-induced changes in vitamin and amino acid biosynthesis and degradation. These results highlight the utility of combining a genome-wide metabolic reconstruction methodology with 16S rRNA sequence-based community profiles to provide insights pertaining to community metabolism. This process also provides a rational means for prioritizing in vivo analysis of prebiotics and medicinal herbs in vivo to test hypotheses related to therapeutic potential in specific diseases of interest.

Mammalian spermatozoa have a surface covered with glycocalyx, consisting of heterogeneous glycoproteins and glycolipids. This complexity arises from diverse monosaccharides, distinct linkages, various isomeric glycans, branching levels, and saccharide sequences. The glycocalyx is synthesized by spermatozoa developing in the testis, and its subsequent alterations during their transit through the epididymis are a critical process for the sperm acquisition of fertilizing ability. In this study, we performed detailed analysis of the glycocalyx on the sperm surface of bull spermatozoa in relation to individual parts of the epididymis using a wide range (24) of lectins with specific carbohydrate binding preferences. Fluorescence analysis of intact sperm isolated from the bull epididymides was complemented by Western blot detection of protein extracts from the sperm plasma membrane fractions. Our experimental results revealed predominant sequential modification of bull sperm glycans with N‐acetyllactosamine (LacNAc), followed by subsequent sialylation and fucosylation in a highly specific manner. Additionally, variations in the lectin detection on the sperm surface may indicate the acquisition or release of glycans or glycoproteins. Our study is the first to provide a complex analysis of the bull sperm glycocalyx modification during epididymal maturation. [ABSTRACT FROM AUTHOR]

Glycan-based scaffolds are unique in their high specificity, versatility, low immunogenicity, and ability to mimic natural carbohydrates, making them attractive candidates for use in cancer treatment. These scaffolds are made up of glycans, which are biopolymers with well biocompatibility in the human body that can be used for drug delivery. The versatility of glycan-based scaffolds allows for the modulation of drug activity and targeted delivery to specific cells or tissues, which increases the potency of drugs and reduces side effects. Despite their promise, there are still technical challenges in the design and production of glycan-based scaffolds, as well as limitations in their therapeutic efficacy and specificity. [ABSTRACT FROM AUTHOR]

This report outlines the case of a child affected by a type of congenital disorder of glycosylation (CDG) known as ALG2-CDG (OMIM 607906), presenting as a congenital myasthenic syndrome (CMS) caused by variants identified in ALG2, which encodes an α1,3-mannosyltransferase (EC 2.4.1.132) involved in the early steps of N-glycosylation. To date, fourteen cases of ALG2-CDG have been documented worldwide. From birth, the child experienced perinatal asphyxia, muscular weakness, feeding difficulties linked to an absence of the sucking reflex, congenital hip dislocation, and hypotonia. Over time, additional complications emerged, such as inspiratory stridor, gastroesophageal reflux, low intake, recurrent seizures, respiratory infections, an inability to maintain the head upright, and a global developmental delay. Whole genome sequencing (WGS) revealed the presence of two ALG2 variants in compound heterozygosity: a novel variant c.1055_1056delinsTGA p.(Ser352Leufs*3) and a variant of uncertain significance (VUS) c.964C>A p.(Pro322Thr). Additional studies, including determination of carbohydrate-deficient transferrin (CDT) revealed a mild type I CDG pattern and the presence of an abnormal transferrin glycoform containing a linear heptasaccharide consisting of one sialic acid, one galactose, one N-acetyl-glucosamine, two mannoses and two N-acetylglucosamines (NeuAc- Gal-GlcNAc-Man2-GlcNAc2), ALG2-CDG diagnostic biomarker, confirming the pathogenicity of these variants. [ABSTRACT FROM AUTHOR]

This review briefly introduces readers to an area where glycomics meets modern oncodiagnostics with a focus on the analysis of sialic acid (Neu5Ac)-terminated structures. We present the biochemical perspective of aberrant sialylation during tumourigenesis and its significance, as well as an analytical perspective on the detection of these structures using different approaches for diagnostic and therapeutic purposes. We also provide a comparison to other established liquid biopsy approaches, and we mathematically define an early-stage cancer based on the overall prognosis and effect of these approaches on the patient's quality of life. Finally, some barriers including regulations and quality of clinical validations data are discussed, and a perspective and major challenges in this area are summarised. [ABSTRACT FROM AUTHOR]

Glycans have attracted much attention in cancer therapeutic strategies, and cell surface proteins and lipids with glycans are known to be altered during the carcinogenic process. However, our understanding of how the glycogenes profile responds to drug stimulation remains incomplete. In this study, we search public databases for Sequence Read Archive data on drug-treated liver cancer cells, with the aim to comprehensively analyze the drug responses of glycogenes via bioinformatic meta-analysis. The study comprised 86 datasets, encompassing eight distinct liver cancer cell lines and 13 different drugs. Differentially expressed genes were quantified, and 399 glycogenes were identified. The glycogenes signature was then analyzed using bioinformatics methodologies. In the Protein-protein interaction network analysis, we identified drug-responsive glycogenes such as Beta-1,4-Galactosyltransferase 1, GDP-Mannose 4,6-Dehydratase, UDP-Glucose Ceramide Glucosyltransferase, and Solute Carrier Family 2 Member 4 as key glycan biomarkers. In the enrichment analysis using the pathway list of glycogenes, the results also demonstrated that drug stimulation resulted in alterations to glycopathway-related genes involved in several processes, namely O-Mannosylation, POMGNT2 Type, Capping, Heparan Sulfate Sulfation, and Glucuronidation pathways. These genes and pathways commonly exhibit variable expression across multiple liver cancer cells in response to the same drug, making them potential targets for new cancer therapies. In addition to their primary roles, drugs may also participate in the regulation of glycans. The insights from this study could pave the way for the development of liver cancer therapies that target the regulation of gene profiles involved in the biosynthesis of glycans. [ABSTRACT FROM AUTHOR]

Introduction: The pathogenic bacterium Helicobacter pylori has evolved glycan-mediated mechanisms to evade host immune defenses. This study tests the hypothesis that genetic disruption of H. pylori glycan biosynthesis alters immune recognition and response by human gastric epithelial cells and monocyte-derived dendritic cells. Methods: To test this hypothesis, human cell lines were challenged with wildtype H. pylori alongside an array of H. pylori glycosylation mutants. The relative levels of immune response were measured via immature dendritic cell maturation and cytokine secretion. Results: Our findings indicate that disruption of lipopolysaccharide biosynthesis diminishes gastric cytokine production, without disrupting dendritic cell recognition and activation. In contrast, variable immune responses were observed in protein glycosylation mutants which prompted us to test the hypothesis that phase variation plays a role in regulating bacterial cell surface glycosylation and subsequent immune recognition. Lewis antigen presentation does not correlate with extent of immune response, while the extent of lipopolysaccharide O-antigen elaboration does. Discussion: The outcomes of this study demonstrate that H. pylori glycans modulate the host immune response. This work provides a foundation to pursue immune-based tailoring of bacterial glycans towards modulating immunogenicity of microbial pathogens. [ABSTRACT FROM AUTHOR]

Glycans are central to information content and regulation in biological systems. These carbohydrate molecules are active either as oligo‐ or polysaccharides, often in the form of glycoconjugates. The monosaccharide entities are joined by glycosidic linkages and stereochemical arrangements are of utmost importance in determining conformation and flexibility of saccharides. The conformational preferences and population distributions at the glycosidic torsion angles φ and ψ have been investigated for O‐methyl glycosides of three disaccharides where the substitution takes place at a secondary alcohol, viz., in α‐l‐Fucp‐(1→3)‐β‐d‐Glcp‐OMe, α‐l‐Fucp‐(1→3)‐α‐d‐Galp‐OMe and α‐d‐Glcp‐(1→4)‐α‐d‐Galp‐OMe, corresponding to disaccharide structural elements present in bacterial polysaccharides. Stereochemical differences at or adjacent to the glycosidic linkage were explored by solution state NMR spectroscopy using one‐dimensional 1H,1H‐NOESY NMR experiments to obtain transglycosidic proton‐proton distances and one‐ and two‐dimensional heteronuclear NMR experiments to obtain 3JCH transglycosidic coupling constants related to torsion angles φ and ψ. Computed effective proton‐proton distances from molecular dynamics (MD) simulations showed excellent agreement to experimentally derived distances for the α‐(1→3)‐linked disaccharides and revealed that for the bimodal distribution at the ψ torsion angle for the α‐(1→4)‐linked disaccharide experiment and simulation were at variance with each other, calling for further force field developments. The MD simulations disclosed a highly intricate inter‐residue hydrogen bonding pattern for the α‐(1→4)‐linked disaccharide, including a nonconventional hydrogen bond between H5′ in the glucosyl residue and O3 in the galactosyl residue, supported by a large downfield 1H NMR chemical shift displacement compared to α‐d‐Glcp‐OMe. Comparison of population distributions of the glycosidic torsion angles φ and ψ in the disaccharide entities to those of corresponding crystal structures highlighted the potential importance of solvation on the preferred conformation. [ABSTRACT FROM AUTHOR]

Lung cancer remains a global health crisis, responsible for significant morbidity and mortality. Late-stage diagnosis often limits treatment options and patient survival. Therefore, identifying reliable and sensitive biomarkers for early detection is crucial. Glycosylation, the addition of glycans to protein/RNA/lipid, is a vital cellular process. Normal glycosylation regulates healthy cell function, while alterations, particularly in fucosylation and sialylation, contribute to lung cancer development and progression. These aberrant glycosylation patterns are associated with processes such as immune modulation, cell migration, proliferation, and cell-cell recognition. Fucosylation, a specific type of glycosylation, is frequently altered in lung cancer, with high levels detected in tumors. Understanding the mechanisms behind this altered fucosylation holds immense potential. It can pave the way for the development of novel therapeutic and diagnostic tools for lung cancer. By analyzing specific fucosylation patterns in bodily fluids, it could lead to early-stage diagnosis. This review delves into the mechanisms of fucosylation in lung cancer initiation and metastasis, proposing promising strategies to target the mechanisms, aiming to inhibit tumor growth and disease progression.

Background and Aims: Cancer-associated venous thromboembolism (VTE) is a frequent complication associated with high mortality in patients with cancer, particularly pancreatic cancer. While biological factors such as coagulation factors released from cancer cells may underlie the mechanisms of cancer-associated VTE, the detailed mechanisms have not been determined. Here, we aimed to determine whether extracellular vesicles carrying a glycan sialyl-Lewisa, known as carbohydrate antigen 19-9 (CA19-9), which is a clinically used serum tumor marker and selectin ligand, are a significant cause of cancer-associated VTE. Methods: Risk factors for cancer-associated VTE were determined using clinical data. EVs derived from CA19-9-deficient or overexpressing pancreatic cancer cells were characterized. The protein levels of coagulation factors on the surface of the EVs were quantified using our newly developed sensitive method. Results: Higher CA19-9 levels in the sera of patients were significantly associated with the occurrence of VTE. Using CA19-9-negative or overexpressing pancreatic cancer cells, we found that EVs derived from these cells interacted with E-selectin of endothelial cells in a CA19-9-dependent manner in cell-based assays and in vitro blood vessel models. EVs derived from cancer cells have higher tissue factor levels on their surfaces, and increased tissue factor activity is induced locally, where CA19-9-positive EVs bind to activated endothelial cells. Conclusion: These results suggest that the binding between CA19-9-positive EVs released from cancer cells and endothelial cell E-selectin explains the increased frequency of VTE in patients with pancreatic cancer.

Oat gum (β-glucan, glycan) belongs to the group of soluble dietary fibers. Due to their important functional properties, cereal β-glucans are used to reduce the level of cholesterol in the blood, alleviate the symptoms of diabetes, lower blood pressure, and prevent cardiovascular and oncological diseases. Sources of β-glucans are yeast, mushrooms, bacteria, barley, oats. The use of secondary resources of barley and oat processing as a source of β-glucans has both economic and environmental significance. Cereal β-glucans consist of β-D-glucose monomers connected by (1,3)- and (1,4)-glycosidic bonds. β-Glucan dissolves well in cold water and is insoluble in ethyl alcohol. It is able to form very viscous solutions at low concentrations. The viscosity of solutions depends on the concentration and molecular weight of β-glucan. The rheological properties of aqueous gum solutions do not depend on the pH of the medium. The presence of NaCl in concentrations of more than 0.5% leads to a decrease in the viscosity of solutions, and the addition of sucrose in the amount of 20-45% increases the viscosity. The technological features of obtaining β-glucans are related to the method of their extraction from natural sources. Extraction of oat gum from oat bran or rolled oats involves alkaline treatment of flour at 50-70 °C and pH 8.0-10.5, removal of starch and proteins, precipitation of oat gum with alcohol and separation by centrifugation. Solubility, degree of extraction, and yield of β-glucan from oats depend on particle size, pretreatment of grain raw materials, and extraction conditions (temperature, pH, extractants). β-Glucan is isolated from the extract by dialysis, ultrafiltration or alcohol precipitation. When using dialysis, highly viscous β-glucan is obtained, but with a lower yield; ultrafiltration and alcohol precipitation allow a higher yield of β-glucan with lower viscosity. Oat gum can be introduced into a wide range of food products to provide new functional properties and improve nutritional quality, includ ng wheat bread, bakery products, cookies, pasta, soft drinks, as a fat substitute in dairy and meat products. It performs the technological functions of a thickener, gelling agent, stabilizer or coating in food systems.

D‐peptide ligands can be screened for therapeutic potency and enzymatic stability using synthetic mirror‐image proteins (D‐proteins), but efficient acquisition of these D‐proteins can be hampered by the need to accomplish their in vitro folding, which often requires the formation of correctly linked disulfide bonds. Here, we report the finding that temporary installation of natural O‐linked‐β‐N‐acetyl‐D‐glucosamine (O‐GlcNAc) groups onto selected D‐serine or D‐threonine residues of the synthetic disulfide‐bonded D‐proteins can facilitate their folding in vitro, and that the natural glycosyl groups can be completely removed from the folded D‐proteins to afford the desired chirally inverted D‐protein targets using naturally occurring O‐GlcNAcase. This approach enabled the efficient chemical syntheses of several important but difficult‐to‐fold D‐proteins incorporating disulfide bonds including the mirror‐image tumor necrosis factor alpha (D‐TNFα) homotrimer and the mirror‐image receptor‐binding domain of the Omicron spike protein (D‐RBD). Our work establishes the use of O‐GlcNAc to facilitate D‐protein synthesis and folding and proves that D‐proteins bearing O‐GlcNAc can be good substrates for naturally occurring O‐GlcNAcase. [ABSTRACT FROM AUTHOR]

Activated microglia-induced inflammation in the hippocampus plays an important role in perioperative neurocognitive disorders. Previous studies have shown that sialic acid-binding immunoglobulin-like lectin 3 (hSiglec-3, ortholog of mouse Siglec-E) engagement in microglia and its glycan ligands on neurons contributes to inflammatory homeostasis through an endogenous negative regulation pathway. This study aimed to explore whether the glycan ligand alteration on neurons plays a role in sevoflurane-induced perioperative neurocognitive disorders. This study's data has shown that a slight Siglec-E ligands' expression decrease does not induce inflammation homeostasis disruption. We also demonstrated that the ligand level on neurons was decreased with age, and the reduced Siglec-E ligand expression on neurons caused via sevoflurane was induced by neuraminidase 1. Furthermore, this study has shown that the Siglec-E ligand expression decline caused by age and sevoflurane treatment could decrease the ligands' level, thus leading to inflammatory homeostasis disruption. This research provided a novel mechanism for perioperative neurocognitive disorder susceptibility in the elderly. [ABSTRACT FROM AUTHOR]

Dendritic cell (DC) subsets play a crucial role in shaping anti‐tumour immunity. Cancer escapes from the control immune system by hijacking DC functions. Yet, bases for such subversion are only partially understood. Tumour cells display aberrant glycan motifs on surface glycoproteins and glycolipids. Such carbohydrate patterns can be sensed by DCs through C‐type lectin receptors (CLRs) that are critical to shape and orientate immune responses. We recently demonstrated that melanoma tumour cells harboured an aberrant 'glyco‐code,' and that circulating and tumour‐infiltrating DCs from melanoma patients displayed major perturbations in their CLR profiles. To decipher whether melanoma, through aberrant glycan patterns, may exploit CLR pathways to mislead DCs and evade immune control, we explored the impact of glycan motifs aberrantly found in melanoma (neoglycoproteins [NeoGP] functionalised with Gal, Man, GalNAc, s‐Tn, fucose [Fuc] and GlcNAc residues) on features of human DC subsets (cDC2s, cDC1s and pDCs). We examined the ability of glycans to bind to purified DCs, and assessed their impact on DC basal properties and functional features using flow cytometry, confocal microscopy and multiplex secreted protein analysis. DC subsets differentially bound and internalised NeoGP depending on the nature of the glycan. Strikingly, Fuc directly remodelled the expression of activation markers and immune checkpoints, as well as the cytokine/chemokine secretion profile of DC subsets. NeoGP interfered with Toll like receptor (TLR)‐signalling and pre‐conditioned DCs to exhibit an altered response to subsequent TLR stimulation, dampening antitumor mediators while triggering pro‐tumoral factors. We further demonstrated that DC subsets can bind NeoGP through CLRs, and identified GalNAc/MGL and s‐Tn/ C‐type lectin‐like receptor 2 (CLEC2) as potential candidates. Moreover, DC dysfunction induced by tumour‐associated carbohydrate molecules may be reversed by interfering with the glycan/CLR axis. These findings revealed the glycan/CLR axis as a promising checkpoint to exploit in order to reshape potent antitumor immunity while impeding immunosuppressive pathways triggered by aberrant tumour glycosylation patterns. This may rescue DCs from tumour hijacking and improve clinical success in cancer patients. [ABSTRACT FROM AUTHOR]

Simple Summary: The intensive poultry production system faces production challenges like pathogenic infections, poor performance, and bird welfare. The use of antibiotics has been reduced due to regulations and increasing antimicrobial resistance, promoting research into viable alternatives. Precision glycans represent an alternative that modulates the gut microbial community and its metabolic functions. This study compares birds fed precision glycan-supplemented and non-supplemented diets in a commercial broiler farm. We report major alterations in microbiota across caecum, ileum, and ileum mucosa gut sections. The treated birds also showed better intestinal morphology and higher weight gain with an improvement in feed efficiency and disease resistance. The poultry industry contributes significantly to the global meat industry but faces many production challenges like high-density housing, welfare issues, and pathogenic infections. While antibiotics have commonly been used to treat many of these issues, they are being removed from poultry production globally due to increased microbial resistance. Precision glycans offer a viable alternative to antibiotics by modulating microbial metabolic pathways. In this study, we investigated the effects of precision glycan supplementation on productivity and gut microbiota in broilers. The experiment was conducted in a commercial setting using 32,400 male Ross chickens randomly divided into three sheds with 10,800 birds each. One shed with 12 pen replicates of 900 birds was used as control, while the other two with an equal number of replicates and birds were assigned to precision glycan supplementation. The treatment significantly improved the average daily weight gain and feed conversion ratio, with a significant modification in the abundance of several bacterial taxa in the caecum, ileum, and ileum mucosa microbial communities. There was increased richness and diversity in the caecum, with a reduction in Proteobacteria and an increase in Firmicutes. Richness remained unchanged in the ileum, with an increase in diversity and reduction in pathogenic genera like Clostridium and Escherichia-Shigella. Ileum mucosa showed a lower abundance of mucin degraders and an increased presence of next-generation probiotics. Supplemented birds showed a high level of disease resistance when the farm experienced an outbreak of infectious bronchitis, evidenced by lower mortality. Histological analysis confirmed improvements in the ileum and liver health, where the precision glycan supplementation reduced the area of congested sinusoids compared to the control group in the liver and significantly improved ileum intestinal morphology by increasing crypt depth and surface area. These results collectively suggest that precision glycans offer substantial benefits in poultry production by improving productivity, gut health, and disease resistance. [ABSTRACT FROM AUTHOR]

The presence of NHAc groups in the substrates (both glycosyl donors and acceptors) significantly reduced the reactivity of glycosylation. This decrease was attributed to the NHAc groups forming intermolecular hydrogen bonds by the NHAc groups, thereby reducing molecular mobility. Hence, a diacetyl strategy involving the temporary conversion of NHAc to diacetyl imide (NAc2) was developed for the synthesis of NHAc-containing glycans. This strategy has two significant advantages for oligosaccharide synthesis. The NAc2 protection of NHAc substantially enhances the rate of glycosylation reactions, resulting in improved yields. Moreover, NAc2 can be readily reverted to NHAc by the simple removal of one acetyl group under mild basic conditions, obviating the necessity for treating the polar amino group. We have achieved the efficient synthesis of oligosaccharides containing GlcNHAc and N-glycans containing sialic acid using the diacetyl strategy. [ABSTRACT FROM AUTHOR]

Plant cell walls are complex structures mainly made up of carbohydrate and phenolic polymers. In addition to their structural roles, cell walls function as external barriers against pathogens and are also reservoirs of glycan structures that can be perceived by plant receptors, activating Pattern-Triggered Immunity (PTI). Since these PTI-active glycans are usually released upon plant cell wall degradation, they are classified as Damage Associated Molecular Patterns (DAMPs). Identification of DAMPs imply their extraction from plant cell walls by using multistep methodologies and hazardous chemicals. Subcritical water extraction (SWE) has been shown to be an environmentally sustainable alternative and a simplified methodology for the generation of glycan-enriched fractions from different cell wall sources, since it only involves the use of water. Starting from Equisetum arvense cell walls, we have explored two different SWE sequential extractions (isothermal at 160 ºC and using a ramp of temperature from 100 to 160 ºC) to obtain glycans-enriched fractions, and we have compared them with those generated with a standard chemical-based wall extraction. We obtained SWE fractions enriched in pectins that triggered PTI hallmarks in Arabidopsis thaliana such as calcium influxes, reactive oxygen species production, phosphorylation of mitogen activated protein kinases and overexpression of immune-related genes. Notably, application of selected SWE fractions to pepper plants enhanced their disease resistance against the fungal pathogen Sclerotinia sclerotiorum. These data support the potential of SWE technology in extracting PTI-active fractions from plant cell wall biomass containing DAMPs and the use of SWE fractions in sustainable crop production. Key message: Subcritical water extraction of Equisetum arvense cell wall generates glycan-enriched fractions that contain Damage Associated Molecular Patterns (DAMPs) that trigger plant immune responses and disease resistance. [ABSTRACT FROM AUTHOR]

This report outlines the case of a child affected by a type of congenital disorder of glycosylation (CDG) known as ALG2-CDG (OMIM 607906), presenting as a congenital myasthenic syndrome (CMS) caused by variants identified in ALG2, which encodes an α1,3-mannosyltransferase (EC 2.4.1.132) involved in the early steps of N-glycosylation. To date, fourteen cases of ALG2-CDG have been documented worldwide. From birth, the child experienced perinatal asphyxia, muscular weakness, feeding difficulties linked to an absence of the sucking reflex, congenital hip dislocation, and hypotonia. Over time, additional complications emerged, such as inspiratory stridor, gastroesophageal reflux, low intake, recurrent seizures, respiratory infections, an inability to maintain the head upright, and a global developmental delay. Whole genome sequencing (WGS) revealed the presence of two ALG2 variants in compound heterozygosity: a novel variant c.1055_1056delinsTGA p.(Ser352Leufs*3) and a variant of uncertain significance (VUS) c.964C>A p.(Pro322Thr). Additional studies, including determination of carbohydrate-deficient transferrin (CDT) revealed a mild type I CDG pattern and the presence of an abnormal transferrin glycoform containing a linear heptasaccharide consisting of one sialic acid, one galactose, one N-acetyl-glucosamine, two mannoses and two N-acetylglucosamines (NeuAc-Gal-GlcNAc-Man2-GlcNAc2), ALG2-CDG diagnostic biomarker, confirming the pathogenicity of these variants.

IntroductionThe pathogenic bacterium Helicobacter pylori has evolved glycan-mediated mechanisms to evade host immune defenses. This study tests the hypothesis that genetic disruption of H. pylori glycan biosynthesis alters immune recognition and response by human gastric epithelial cells and monocyte-derived dendritic cells.MethodsTo test this hypothesis, human cell lines were challenged with wildtype H. pylori alongside an array of H. pylori glycosylation mutants. The relative levels of immune response were measured via immature dendritic cell maturation and cytokine secretion.ResultsOur findings indicate that disruption of lipopolysaccharide biosynthesis diminishes gastric cytokine production, without disrupting dendritic cell recognition and activation. In contrast, variable immune responses were observed in protein glycosylation mutants which prompted us to test the hypothesis that phase variation plays a role in regulating bacterial cell surface glycosylation and subsequent immune recognition. Lewis antigen presentation does not correlate with extent of immune response, while the extent of lipopolysaccharide O-antigen elaboration does.DiscussionThe outcomes of this study demonstrate that H. pylori glycans modulate the host immune response. This work provides a foundation to pursue immune-based tailoring of bacterial glycans towards modulating immunogenicity of microbial pathogens.

Abstract Objective Galectins are sugar-binding proteins that participate in many biological processes, such as immunity, by regulating host immune cells and their direct interaction with pathogens. They are involved in mediating infection by Schistosoma mansoni, a parasitic trematode that causes schistosomiasis. However, their direct effects on schistosomes have not been investigated. Results We found that galectin-2 recognizes S. mansoni glycoconjugates and investigated whether galectin-1, 2, and 3 can directly affect S. mansoni in vitro. Adult S. mansoni were treated with recombinant galectin-1, 2, and 3 proteins or praziquantel, a positive control. Treatment with galectin-1, 2, and 3 had no significant effect on S. mansoni motility, and no other differences were observed under a stereoscopic microscope. Hence, galectin-1, 2, and 3 may have a little direct effect on S. mansoni. However, they might play a role in the infection in vivo via the modulation of the host immune response or secretory molecules from S. mansoni. To the best of our knowledge, this is the first study to investigate the direct effect of galectins on S. mansoni and helps in understanding the roles of galectins in S. mansoni infection in vivo.

Abstract Extracellular vesicles (EVs) have considerable potential as diagnostic, prognostic, and therapeutic agents, in large part because molecular patterns on the EV surface betray the cell of origin and may also be used to “target” EVs to specific cells. Cancer is associated with alterations to cellular and EV glycosylation patterns, and the surface of EVs is enriched with glycan moieties. Glycoconjugates of EVs play versatile roles in cancer including modulating immune response, affecting tumor cell behavior and site of metastasis and as such, paving the way for the development of innovative diagnostic tools and novel therapies. Entities that recognize specific glycans, such as lectins, may thus be powerful tools to discover and detect novel cancer biomarkers. Indeed, the past decade has seen a constant increase in the number of published articles on lectin-based strategies for the detection of EV glycans. This review explores the roles of EV glycosylation in cancer and cancer-related applications. Furthermore, this review summarizes the potential of lectins and lectin-based methods for screening, targeting, separation, and possible identification of improved biomarkers from the surface of EVs.

Salmonella invades and disrupts gut epithelium integrity, creating an infection-generated electric field that can drive directional migration of macrophages, a process called galvanotaxis. Phagocytosis of bacteria reverses the direction of macrophage galvanotaxis, implicating a bioelectrical mechanism to initiate life-threatening disseminations. The force that drives direction reversal of macrophage galvanotaxis is not understood. One hypothesis is that Salmonella can alter the electrical properties of the macrophages by modifying host cell surface glycan composition, which is supported by the fact that cleavage of surface-exposed sialic acids with a bacterial neuraminidase severely impairs macrophage galvanotaxis, as well as phagocytosis. Here, we utilize N-glycan profiling by nanoLC-chip QTOF mass cytometry to characterize the bacterial neuraminidase-associated compositional shift of the macrophage glycocalyx, which revealed a decrease in sialylated and an increase in fucosylated and high mannose structures. The Salmonella nanH gene, encoding a putative neuraminidase, is required for invasion and internalization in a human colonic epithelial cell infection model. To determine whether NanH is required for the Salmonella infection-dependent direction reversal, we constructed and characterized a nanH deletion mutant and found that NanH is partially required for Salmonella infection in primary murine macrophages. However, compared to wild type Salmonella, infection with the nanH mutant only marginally reduced the cathode-oriented macrophage galvonotaxis, without canceling direction reversal. Together, these findings strongly suggest that while neuraminidase-mediated N-glycan modification impaired both macrophage phagocytosis and galvanotaxis, yet to be defined mechanisms other than NanH may play a more important role in bioelectrical control of macrophage trafficking, which potentially triggers dissemination.

BackgroundGlycoproteomics deals with glycoproteins that are formed by post-translational modification when sugars (like fucose and sialic acid) are attached to protein. Glycosylation of proteins influences function, but whether glycosylation is altered by diet is unknown.ObjectiveTo evaluate the effect of consuming a diet based on the Dietary Guidelines for Americans on circulating glycoproteins that have previously been associated with cardiometabolic diseases.DesignForty-four women, with one or more metabolic syndrome characteristics, completed an 8-week randomized controlled feeding intervention (n = 22) consuming a diet based on the Dietary Guidelines for Americans (DGA 2010); the remaining consumed a 'typical American diet' (TAD, n = 22). Fasting serum samples were obtained at week0 (baseline) and week8 (post-intervention); 17 serum proteins were chosen for targeted analyses. Protein standards and serum samples were analyzed in a UHPLC-MS protocol to determine peptide concentration and their glycan (fucosylation or sialylation) profiles. Data at baseline were used in correlational analyses; change in proteins and glycans following intervention were used in non-parametric analyses.ResultsAt baseline, women with more metabolic syndrome characteristics had more fucosylation (total di-fucosylated proteins: p = 0.045) compared to women with a lesser number of metabolic syndrome characteristics. Dietary refined grain intake was associated with increased total fucosylation (ρ = - 0.530, p

Abstract Background Alpha-Gal syndrome (AGS) is a tick-borne food allergy caused by IgE antibodies against the glycan galactose-alpha-1,3-galactose (α-Gal) present in glycoproteins and glycolipids from mammalian meat. To advance in the diagnosis and treatment of AGS, further research is needed to unravel the molecular and immune mechanisms underlying this syndrome. The objective of this study is the characterization of tick salivary components and proteins with and without α-Gal modifications involved in modulating human immune response against this carbohydrate. Methods Protein and α-Gal content were determined in tick saliva components, and proteins were identified by proteomics analysis of tick saliva fractions. Pathophysiological changes were recorded in the zebrafish (Danio rerio) model after exposure to distinct Ixodes ricinus tick salivary components. Serum samples were collected from zebrafish at day 8 of exposure to determine anti-α-Gal, anti-glycan, and anti-tick saliva protein IgM antibody titers by enzyme-linked immunosorbent assay (ELISA). Results Zebrafish treated with tick saliva and saliva protein fractions combined with non-protein fractions demonstrated significantly higher incidence of hemorrhagic type allergic reactions, abnormal behavioral patterns, or mortality when compared to the phosphate-buffered saline (PBS)-treated control group. The main tick salivary proteins identified in these fractions with possible functional implication in AGS were the secreted protein B7P208-salivary antigen p23 and metalloproteases. Anti-α-Gal and anti-tick salivary gland IgM antibody titers were significantly higher in distinct saliva protein fractions and deglycosylated saliva group when compared with PBS-treated controls. Anti-glycan antibodies showed group-related profiles. Conclusions Results support the hypothesis that tick salivary biomolecules with and without α-Gal modifications are involved in modulating immune response against this carbohydrate. Graphical Abstract

ABSTRACT Many pathogenic Gram-negative bacteria use repeats-in-toxin adhesins for colonization and biofilm formation. In the cholera agent Vibrio cholerae, flagellar-regulated hemagglutinin A (FrhA) enables these functions. Using bioinformatic analysis, a sugar-binding domain was identified in FrhA adjacent to a domain of unknown function. AlphaFold2 indicated the boundaries of both domains to be slightly shorter than previously predicted and assisted in the recognition of the unknown domain as a split immunoglobulin-like fold that can assist in projecting the sugar-binding domain toward its target. The AlphaFold2-predicted structure is in excellent agreement with the molecular envelope obtained from small-angle X-ray scattering analysis of a recombinant construct spanning the sugar-binding and unknown domains. This two-domain construct was probed by glycan micro-array screening and showed binding to mammalian fucosylated glycans, some of which are characteristic erythrocyte markers and intestinal cell epitopes. Isothermal titration calorimetry further showed the construct-bound l-fucose with a Kd of 21 µM. Strikingly, this recombinant protein construct bound and lysed erythrocytes in a concentration-dependent manner, and its hemolytic activity was blocked by the addition of l-fucose. A protein ortholog construct from Aeromonas veronii was also produced and showed a similar glycan-binding pattern, binding affinity, erythrocyte-binding, and hemolytic activities. As demonstrated here with Hep-2 cells, fucose-based inhibitors of this sugar-binding domain can potentially be developed to block colonization by V. cholerae and other pathogenic bacteria that share this adhesin domain.IMPORTANCEThe bacterium, Vibrio cholerae, which causes cholera, uses an adhesion protein to stick to human cells and begin the infection process. One part of this adhesin protein binds to a particular sugar, fucose, on the surface of the target cells. This binding can lead to colonization and killing of the cells by the bacteria. Adding l-fucose to the bacteria before they bind to the human cells can prevent attachment and has promise as a preventative drug to protect against cholera.

IntroductionSince the outbreak of SARS-CoV-2, vaccines have demonstrated their effectiveness in resisting virus infection, reducing severity, and lowering the mortality rate in infected individuals. However, due to the rapid and ongoing mutations of SARS-CoV-2, the protective ability of many available vaccines has been challenged. Therefore, there is an urgent need for vaccines capable of eliciting potent broadly neutralizing antibodies against various SARS-CoV-2 variants.MethodsIn this study, we developed a novel subunit vaccine candidate for SARS-CoV-2 by introducing a series of shielding glycans to the Fc-fused receptor-binding domain (RBD) of the prototypic spike protein. This approach aims to mask non-neutralizing epitopes and focus the immune response on crucial neutralizing epitopes.ResultsAll modified sites were confirmed to be highly glycosylated through mass spectrometry analysis. The binding affinity of the glycan-shielded RBD (gsRBD) to the human ACE2 receptor was comparable to that of the wildtype RBD (wtRBD). Immunizing mice with gsRBD when combined with either Freund’s adjuvant or aluminum adjuvant demonstrated that the introduction of the glycan shield did not compromise the antibody-inducing ability of RBD. Importantly, the gsRBD significantly enhanced the generation of neutralizing antibodies against SARS-CoV-2 pseudoviruses compared to the wtRBD. Notably, it exhibited remarkable protective activity against Beta (B.1.351), Delta (B.1.617.2), and Omicron (B.1.1.529), approximately 3-fold, 7- fold, and 17-fold higher than wtRBD, respectively.DiscussionOur data proved this multiple-epitope masking strategy as an effective approach for highly active vaccine production.

The presence of NHAc groups in the substrates (both glycosyl donors and acceptors) significantly reduced the reactivity of glycosylation. This decrease was attributed to the NHAc groups forming intermolecular hydrogen bonds by the NHAc groups, thereby reducing molecular mobility. Hence, a diacetyl strategy involving the temporary conversion of NHAc to diacetyl imide (NAc2) was developed for the synthesis of NHAc-containing glycans. This strategy has two significant advantages for oligosaccharide synthesis. The NAc2 protection of NHAc substantially enhances the rate of glycosylation reactions, resulting in improved yields. Moreover, NAc2 can be readily reverted to NHAc by the simple removal of one acetyl group under mild basic conditions, obviating the necessity for treating the polar amino group. We have achieved the efficient synthesis of oligosaccharides containing GlcNHAc and N-glycans containing sialic acid using the diacetyl strategy.

Glycans are essential to all scales of biology, with their intricate structures being crucial for their biological functions. The structural complexity of glycans is communicated through simplified and unified visual representations according to the Symbol Nomenclature for Glycans (SNFGs) guidelines adopted by the community. Here, we introduce GlycoDraw, a Python-native implementation for high-throughput generation of high-quality, SNFG-compliant glycan figures with flexible display options. GlycoDraw is released as part of our glycan analysis ecosystem, glycowork, facilitating integration into existing workflows by enabling fully automated annotation of glycan-related figures and thus assisting the analysis of e.g. differential abundance data or glycomics mass spectra. [ABSTRACT FROM AUTHOR]

Background: Alpha-Gal syndrome (AGS) is a tick-borne food allergy caused by IgE antibodies against the glycan galactose-alpha-1,3-galactose (α-Gal) present in glycoproteins and glycolipids from mammalian meat. To advance in the diagnosis and treatment of AGS, further research is needed to unravel the molecular and immune mechanisms underlying this syndrome. The objective of this study is the characterization of tick salivary components and proteins with and without α-Gal modifications involved in modulating human immune response against this carbohydrate. Methods: Protein and α-Gal content were determined in tick saliva components, and proteins were identified by proteomics analysis of tick saliva fractions. Pathophysiological changes were recorded in the zebrafish (Danio rerio) model after exposure to distinct Ixodes ricinus tick salivary components. Serum samples were collected from zebrafish at day 8 of exposure to determine anti-α-Gal, anti-glycan, and anti-tick saliva protein IgM antibody titers by enzyme-linked immunosorbent assay (ELISA). Results: Zebrafish treated with tick saliva and saliva protein fractions combined with non-protein fractions demonstrated significantly higher incidence of hemorrhagic type allergic reactions, abnormal behavioral patterns, or mortality when compared to the phosphate-buffered saline (PBS)-treated control group. The main tick salivary proteins identified in these fractions with possible functional implication in AGS were the secreted protein B7P208-salivary antigen p23 and metalloproteases. Anti-α-Gal and anti-tick salivary gland IgM antibody titers were significantly higher in distinct saliva protein fractions and deglycosylated saliva group when compared with PBS-treated controls. Anti-glycan antibodies showed group-related profiles. Conclusions: Results support the hypothesis that tick salivary biomolecules with and without α-Gal modifications are involved in modulating immune response against this carbohydrate. [ABSTRACT FROM AUTHOR]

Objective: Galectins are sugar-binding proteins that participate in many biological processes, such as immunity, by regulating host immune cells and their direct interaction with pathogens. They are involved in mediating infection by Schistosoma mansoni, a parasitic trematode that causes schistosomiasis. However, their direct effects on schistosomes have not been investigated. Results: We found that galectin-2 recognizes S. mansoni glycoconjugates and investigated whether galectin-1, 2, and 3 can directly affect S. mansoni in vitro. Adult S. mansoni were treated with recombinant galectin-1, 2, and 3 proteins or praziquantel, a positive control. Treatment with galectin-1, 2, and 3 had no significant effect on S. mansoni motility, and no other differences were observed under a stereoscopic microscope. Hence, galectin-1, 2, and 3 may have a little direct effect on S. mansoni. However, they might play a role in the infection in vivo via the modulation of the host immune response or secretory molecules from S. mansoni. To the best of our knowledge, this is the first study to investigate the direct effect of galectins on S. mansoni and helps in understanding the roles of galectins in S. mansoni infection in vivo. [ABSTRACT FROM AUTHOR]

The advent of high-throughput sequencing technologies has facilitated the profiling of glycosylation genes at a single-cell level in complex biological systems, but the significance of these gene signatures to the composition of the glycocalyx remains ambiguous. Here, we used lectin microarrays to characterize the composition of cell surface glycans in human and mouse corneas and determine its relationship to single-cell transcriptomic data. Our results identify a series of cell surface glycan signatures that are unique to the different cell types of the human cornea and that correlate, to a certain extent, with the transcriptional expression of glycosylation genes. These include pathways involved in the biosynthesis of O-glycans in epithelial cells and core fucose on stromal and endothelial cell surfaces. Moreover, we show that human and mouse corneas display some structural differences in terms of cell surface glycan composition. These results could provide insights into the specialized function of individual cell types in the cornea and foster the identification of novel cornea-specific biomarkers. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) have considerable potential as diagnostic, prognostic, and therapeutic agents, in large part because molecular patterns on the EV surface betray the cell of origin and may also be used to "target" EVs to specific cells. Cancer is associated with alterations to cellular and EV glycosylation patterns, and the surface of EVs is enriched with glycan moieties. Glycoconjugates of EVs play versatile roles in cancer including modulating immune response, affecting tumor cell behavior and site of metastasis and as such, paving the way for the development of innovative diagnostic tools and novel therapies. Entities that recognize specific glycans, such as lectins, may thus be powerful tools to discover and detect novel cancer biomarkers. Indeed, the past decade has seen a constant increase in the number of published articles on lectin-based strategies for the detection of EV glycans. This review explores the roles of EV glycosylation in cancer and cancer-related applications. Furthermore, this review summarizes the potential of lectins and lectin-based methods for screening, targeting, separation, and possible identification of improved biomarkers from the surface of EVs. [ABSTRACT FROM AUTHOR]

The human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer mediates entry into host cells by binding receptors, CD4 and CCR5/CXCR4, and fusing the viral and cell membranes. In infected cells, cleavage of the gp160 Env precursor yields the mature Env trimer, with gp120 exterior and gp41 transmembrane Env subunits. Env cleavage stabilizes the State-1 conformation, which is the major target for broadly neutralizing antibodies, and decreases the spontaneous sampling of more open Env conformations that expose epitopes for poorly neutralizing antibodies. During HIV-1 entry into cells, CD4 binding drives the metastable Env from a pretriggered (State-1) conformation into more "open," lower-energy states. Here, we report that changes in two dissimilar elements of the HIV-1 Env trimer, namely particular gp120 glycans and the gp41 fusion peptide-proximal region (FPPR), can independently modulate the stability of State 1. Individual deletion of several gp120 glycans destabilized State 1, whereas removal of a V1 glycan resulted in phenotypes indicative of a more stable pretriggered Env conformation. Likewise, some alterations of the gp41 FPPR decreased the level of spontaneous shedding of gp120 from the Env trimer and stabilized the pretriggered State-1 Env conformation. State-1-stabilizing changes were additive and could suppress the phenotypes associated with State-1-destabilizing alterations in Env. Our results support a model in which multiple protein and carbohydrate elements of the HIV-1 Env trimer additively contribute to the stability of the pretriggered (State-1) conformation. The Env modifications identified in this study will assist efforts to characterize the structure and immunogenicity of the metastable State-1 conformation. IMPORTANCE: The elicitation of antibodies that neutralize multiple strains of HIV-1 is an elusive goal that has frustrated the development of an effective vaccine. The pretriggered shape of the HIV-1 envelope glycoprotein (Env) spike on the virus surface is the major target for such broadly neutralizing antibodies. The "closed" pretriggered Env shape resists the binding of most antibodies but is unstable and often assumes "open" shapes that elicit ineffective antibodies. We identified particular changes in both the protein and the sugar components of the Env trimer that stabilize the pretriggered shape. Combinations of these changes were even more effective at stabilizing the pretriggered Env than the individual changes. Stabilizing changes in Env could counteract the effect of Env changes that destabilize the pretriggered shape. Locking Env in its pretriggered shape will assist efforts to understand the Env spike on the virus and to incorporate this shape into vaccines. [ABSTRACT FROM AUTHOR]

Abstract Carrying the apolipoprotein E (ApoE) Ɛ4 allele is associated with an increased risk of cerebral amyloidosis and late-onset Alzheimer’s disease, but the degree to which apoE glycosylation affects its development is not clear. In a previous pilot study, we identified distinct total and secondary isoform-specific cerebral spinal fluid (CSF) apoE glycosylation profiles, with the E4 isoform having the lowest glycosylation percentage (E2 > E3 > E4). In this work, we extend the analysis to a larger cohort of individuals (n = 106), utilizing matched plasma and CSF samples with clinical measures of AD biomarkers. The results confirm the isoform-specific glycosylation of apoE in CSF, resulting from secondary CSF apoE glycosylation patterns. CSF apoE glycosylation percentages positively correlated with CSF Aβ42 levels (r = 0.53, p E3 and increased binding affinity to heparin. These results indicate that apoE glycosylation has a new and important role in influencing brain Aβ metabolism and can be a potential target of treatment.

Abstract Purpose Thyroid cancer recurrence following curative thyroidectomy is associated with increased morbidity and mortality, but current surveillance strategies are inadequate for early detection. Prior studies indicate that tissue glycosylation is altered in thyroid cancer, but the utility of serum glycosylation in thyroid cancer surveillance remains unexplored. We therefore assessed the potential utility of altered serum glycomic profile as a tumor‐specific target for disease surveillance in recurrent thyroid cancer. Experimental design We employed banked serum samples from patients with recurrent thyroid cancer post thyroidectomy and healthy controls. N‐glycans were enzymatically released from serum glycoproteins, labeled via permethylation, and analyzed by MALDI‐TOF mass spectrometry. Global level and specific subtypes of glycan structures were compared between patients and controls. Results We evaluated 28 independent samples from 13 patients with cancer recurrence and 15 healthy controls. Global features of glycosylation, including N‐glycan class and terminal glycan modifications were similar between groups, but three of 35 individual glycans showed significant differences. The three glycans were biosynthetically related biantennary core fucosylated N‐glycans that only varied by the degree of galactosylation (G0F, G1F, and G2F; G: galactose, F: fucose). The ratio of G0F:G1F that captures reduced galactosylation was observed in patients samples but not in healthy controls (p = 0.004) and predicted thyroid cancer recurrence (AUC = 0.82, CI 95% = 0.64–0.99). Conclusions Altered N‐glycomic profile was associated with thyroid cancer recurrence. This serum‐based biomarker would be useful as an effective surveillance tool to improve the care and prognosis of thyroid cancer after prospective validation.