Search Results (457)

Peach (Prunus persica) leaves, usually discarded in traditional Chinese medicine, were explored as a source of laxative agents. Using zebrafish larvae for bioactivity-guided fractionation, we isolated a single active flavanone that was identified by NMR and HR-MS as Sakuranetin. In vivo assays demonstrated that Sakuranetin (10–25 µM) accelerated intestinal transit in a dose-dependent fashion; at 25 µM, 64.8% of the fluorescent intestinal content was expelled. Untargeted LC-MS metabolomic analysis revealed significant perturbations in serine biosynthesis and N-glycan precursor biosynthesis, suggesting energetic rewiring of enterocytes. RNA-Seq analysis highlighted gnat1 as the most responsive gene, and molecular docking predicted a stable Sakuranetin–Gnat1 complex with a binding free energy of −8.7 kcal/mol. Concurrent down-regulation of rho transcripts indicated suppression of inflammatory signaling that often accompanies constipation. Our findings identified Sakuranetin as a potent promoter of gut motility and position the otherwise wasted peach leaves as an untapped botanical resource for developing anti-constipation therapeutics. Full article

Norovirus–bacterial interactions influence viral replication and immune responses, yet the molecular details that mediate binding of these viruses to commensal bacteria are unknown. Studies with other enteric viruses have revealed that LPS and other lipid/carbohydrate structures facilitate virus–bacterial interactions, and it has also been shown that human noroviruses (HuNoVs) can interact with histo-blood group antigen (HBGA)-like compounds on the surface of bacterial cells. Based on these findings, this study hypothesized that carbohydrate-based compounds were the ligands that facilitated binding of both human and murine noroviruses (MNV) to bacteria. Using glycan microarrays, competitive inhibition assays, and a panel of bacterial mutants, the project assessed the influence of specific glycans on viral attachment to bacteria. Protein-based interactions were also examined. The results supported previous work which demonstrated that HuNoVs strongly bind HBGA-like glycans, while MNV displayed distinct binding to other glycans including aminoglycosides and fucosylated structures. Ultimately, this work demonstrates that HuNoVs have more limited binding requirements for bacterial attachment compared to MNV, and the MNV binding to bacteria may involve both specific structures as well as electrostatic interactions. Given the importance of commensal bacteria during viral infection, defining the molecular mechanisms that mediate virus–bacteria interactions is critical for understanding infection dynamics and may be useful in the development of disease therapeutics and novel technologies for viral detection from food and environmental sources. Full article

Multivalent lectin–glycan interactions (MLGIs) are vital for viral infection, cell-cell communication and regulation of immune responses. Their structural and biophysical data are thus important, not only for providing insights into their underlying mechanisms but also for designing potent glycoconjugate therapeutics against target MLGIs. However, such information remains to be limited for some important MLGIs, significantly restricting the research progress. We have recently demonstrated that functional nanoparticles, including ∼4 nm quantum dots and varying sized gold nanoparticles (GNPs), densely glycosylated with various natural mono- and oligo- saccharides, are powerful biophysical probes for MLGIs. Using two important viral receptors, DC-SIGN and DC-SIGNR (together denoted as DC-SIGN/R hereafter), as model multimeric lectins, we have shown that α-mannose and α-manno-α-1,2-biose (abbreviated as Man and DiMan, respectively) coated GNPs not only can provide sensitive measurement of MLGI affinities but also reveal critical structural information (e.g., binding site orientation and mode) which are important for MLGI targeting. In this study, we produced mannuronic acid (ManA) coated GNPs (GNP-ManA) of two different sizes to probe the effect of glycan modification on their MLGI affinity and antiviral property. Using our recently developed GNP fluorescence quenching assay, we find that GNP-ManA binds effectively to both DC-SIGN/R and increasing the size of GNP significantly enhances their MLGI affinity. Consistent with this, increasing the GNP size also significantly enhances their ability to block DC-SIGN/R-augmented virus entry into host cells. Particularly, ManA coated 13 nm GNP potently block Ebola virus glycoprotein-driven entry into DC-SIGN/R-expressing cells with sub-nM levels of EC₅₀. Our findings suggest that GNP-ManA probes can act as a useful tool to quantify the characteristics of MLGIs, where increasing the GNP scaffold size substantially enhances their MLGI affinity and antiviral potency. Full article

Galectin-3 (Gal-3) is a β-galactoside-binding lectin that mediates diverse signaling events in multiple cell types, including immune cells. It is also a prognostic indicator for multiple clinically important disorders, including cardiovascular disease. Gal-3 binds to cell surface glycans to form lattices that modulate surface receptor signaling and internalization. However, the tissue-specific regulation of Gal-3 surface expression remains poorly understood. Here, we review evidence for the involvement of Gal-3 in cell surface signaling, intranuclear events, and intracellular trafficking. Our focus will be on the O-GlcNAc modification as a regulator of Gal-3 biosynthesis, non-canonical secretion, and recycling. We argue that the nutrient-driven cytoplasmic hexosamine biosynthetic pathway (HBP) and endomembrane transport mechanisms generate unique pools of nucleotide sugars. The differing levels of nucleotide sugars in the cytosol, endoplasmic reticulum (ER), and Golgi apparatus generate differential thresholds for the responsiveness of O-GlcNAc cycling, N- and O-linked glycan synthesis/branching, and glycolipid synthesis. By regulating Gal-3 synthesis and non-canonical secretion, O-GlcNAc cycling may serve as a nexus constraining Gal-3 cell surface expression and lattice formation. This homeostatic feedback mechanism would be critical under conditions where extensive glycan synthesis and branching in the endomembrane system and on the cell surface are maintained by elevated hexosamine synthesis. Thus, O-GlcNAc cycling and Gal-3 synergize to regulate Gal-3 secretion and influence cellular signaling. In humans, Gal-3 serves as an early-stage prognostic indicator for heart disease, kidney disease, viral infection, autoimmune disease, and neurodegenerative disorders. Since O-GlcNAc cycling has also been linked to these pathologic states, exploring the interconnections between O-GlcNAc cycling and Gal-3 expression and synthesis is likely to emerge as an exciting area of research. Full article

Fungal cell walls, composed of polysaccharides and proteins, play critical roles in adaptation, cell division, and protection against environmental stress. Their polyglucan components are continuously remodeled by various types of glycosyl hydrolases (GHs) and transferases (GTs). In Saccharomyces cerevisiae and other ascomycetes, enzymes of the Dfg5 subfamily, which belong as GTs to the GH76 family, cleave an α1,4 linkage between glucosamine and mannose to facilitate covalent linkage of GPI-anchored proteins to the cell wall’s polyglucans. In contrast, the functions of other fungal GH76 subfamilies are not understood. We characterized CtGH76 from the sordariomycete Chaetomium thermophilum, a member of the Fungi/Bacteria-mixed GH76 subfamily, revealing conserved structural features and functional divergence within the GH76 family. Notably, our structural characterization by X-ray crystallography combined with glycan fragment screening indicated that CtGH76 can recognize GPI-anchors like members of the Dfg5 subfamily but shows a broader promiscuity toward other glycans with central α1,6-mannobiose motifs due to the presence of an elongated glycan-binding canyon. These findings provide new insights into GH76 enzyme diversity and fungal cell wall maturation. Full article

Fucosylation plays a fundamental role in maintaining cellular functions and biological processes across all animals. As a form of glycosylation, it involves the biochemical addition of fucose, a six-carbon monosaccharide, to biological molecules like lipids, proteins, and glycan chains. This modification is essential for optimizing cellular interactions required for receptor-ligand binding, cell adhesion, immune responses, and development. Disruptions in cellular fucose synthesis or in the mechanisms enabling its transfer to other molecules have been linked to human disease. Inherited defects in the fucosylation pathway are rare, with about thirty patients described. Through genome-wide association studies (GWAS), variants in fucosylation pathway genes have been associated with complex diseases such as glaucoma and stroke, and somatic mutations are often found in cancers. Recent studies have applied targeted genetic animal models to elucidate the mechanisms through which disruptions in fucosylation contribute to disease pathogenesis and progression. Key focus areas include GDP-fucose synthesis through de novo or salvage pathways, GDP-fucose transport into the Golgi and endoplasmic reticulum (ER), and its transfer by fucosyltransferases (FUTs) or protein O-fucosyltransferases (POFUTs) onto acceptor molecules. Loss or gain of function fucosylation gene mutations in animal models such as mice, zebrafish, and invertebrates have provided insights into some fucosylation disease pathogenesis. This review aims to bring together these findings, summarizing key insights from existing animal studies to possibly infer fucosylation disease mechanisms in humans. Full article

Momordica balsamina, a plant traditionally used in African medicine, contains a 30 kDa protein, MoMo30, previously identified by our group as an anti-HIV agent that binds glycan residues on the gp120 envelope protein, thereby acting as an entry inhibitor. In this study, we investigated whether prolonged exposure to MoMo30 exerts selective pressure on HIV-1 and induces mutations in the viral envelope (env) gene. T-lymphocyte cells were infected with HIV-1_NL4-3 and continuously treated with MoMo30 over a 24-day period. Viral RNA was isolated at regular intervals, and env genes were sequenced using the Illumina platform. RNA sequence variant calling was performed using iVar, which uses a frequency-based binomial test with a default allele frequency threshold of 3% and a minimum base quality of 20 and applies Bonferroni correction for multiple testing. The infectivity of the MoMo30-exposed virus was assessed using MAGI-CXCR4 cells, visualized by β-galactosidase staining, and compared to untreated controls. Statistical significance was determined via two-way ANOVA. MoMo30-treated HIV-1 exhibited multiple detrimental mutations in gp120 and gp41, including missense, nonsense, and frameshift changes. Notably, 32% of N-linked glycosylation sites were deleted in the treated virus, while no such changes were observed in controls. Functionally, the MoMo30-treated virus demonstrated a sixfold reduction in infectivity compared to untreated HIV-1_NL4-3. These findings suggest that MoMo30 imposes genetic pressure on HIV-1_NL4-3, selecting for mutations that reduce viral fitness. Full article

Glycans on the surface of all immune cells are the product of diverse post-translational modifications (glycosylation) that affect almost all proteins and possess enormous structural heterogeneity. Their bioinformational content is decoded by glycan-binding proteins (lectins, GBPs), such as C-type lectins, including selectins, galectins, and Siglecs. Glycans located on the surface of immune cells are involved in many immunological processes through interactions with GBPs. Lectins recognize changes in the glycan epitopes; distinguish among host (self), microbial (non-self), and tumor (modified self) antigens; and consequently regulate immune responses. Understanding GBP–glycan interactions accelerates the development of glycan-targeted therapeutics in severe diseases, including inflammatory and autoimmune diseases and cancer. This review will discuss N- and O-glycosylations and glycosyltransferases involved in the biosynthesis of carbohydrate epitopes and address how interactions between glycan epitopes and GBPs are crucial in immune responses. The pivotal role of the glycan antigen tetrasaccharide sialyl Lewis x in mediating immune and tumor cell trafficking into the extravascular site will be discussed. Next, the role of glycans in modulating bacterial, fungal, viral, and parasitic infections and cancer will be surveyed. Finally, the role of glycosylation in antibodies and carbohydrate vaccines will be analyzed. Full article

Galectin-12, a member of the galectin family of glycan-binding proteins, exhibits restricted tissue distribution, primarily in adipocytes and sebocytes. In sebocytes, it modulates the cell cycle, influences lipid metabolism through interactions with peroxisome proliferator-activated receptor γ (PPARγ), and exerts immunomodulatory functions by activating immune signaling pathways. Dysregulation of sebocyte homeostasis, lipid metabolism, and immune responses contributes to the pathogenesis of a number of skin diseases, underscoring the therapeutic potential of targeting galectin-12. The review summarizes and discusses current developments in the field to foster future research in this important but underexplored galectin. Full article

Aberrant glycosylation, especially sialylation, on cell surface is often associated with cancer progression and immunosuppression. Over-sialylation of stage-specific embryonic antigen-4 (SSEA-4) to generate disialylGb5 (DSGb5) was reported to trigger Siglec-7 recognition and suppress NK-mediated target killing. In this study, efficient chemo-enzymatic and programmable one-pot methods were explored for the synthesis of DSGb5 and related sialosides for assembly of glycan microarrays and evaluation of binding specificity toward Siglecs-7, 9, 10, and 15 associated with immune checkpoint inhibition. The result showed weak binding of DSGb5 to these Siglecs; however, a truncated glycolyl glycan was identified to bind Siglec-10 strongly with a dissociation constant of 50 nM and exhibited a significant inhibition of Siglec-10 interacting with breast cancer cells. Full article

Metastatic melanoma is an aggressive skin cancer with a five-year survival rate of only 35%. Despite recent advances in immunotherapy, there is still an urgent need for the development of innovative therapeutic approaches to improve clinical outcomes of patients with metastatic melanoma. Prior research from our laboratory revealed that loss of the I-branching enzyme β1,6 N-acetylglucosaminyltransferase 2 (GCNT2), with consequent substitution of melanoma surface I-branched poly-N-acetyllactosamines (poly-LacNAcs) with i-linear poly-LacNAcs, is implicated in driving melanoma metastasis. In the current study, we explored the role of galectin-3 (Gal-3), a lectin that avidly binds surface poly-LacNAcs, in dictating melanoma aggressive behavior. Our results show that Gal-3 favors binding to i-linear poly-LacNAcs, while enforced GCNT2/I-branching disrupts this interaction, thereby suppressing Gal-3-dependent malignant characteristics, including extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway activation, BCL2 expression, cell proliferation, and migration. This report establishes the crucial role of extracellular Gal-3 interactions with i-linear glycans in promoting melanoma cell aggressiveness, placing GCNT2 as a tumor suppressor protein and suggesting both extracellular Gal-3 and i-linear glycans as potential therapeutic targets for metastatic melanoma. Full article

Background: N-glycosylation is a post-translational modification involving the attachment of oligosaccharides to proteins and is known to influence immunoglobulin G (IgG) effector functions and even antigen binding. IgG contains an evolutionarily conserved N-glycosylation site in its fragment crystallizable (Fc) region, while during V-D-J recombination and somatic hypermutation processes it can also obtain N-glycosylation sites in its antigen binding fragment (Fab). Our previous study demonstrated altered IgG N-glycosylation in children at type 1 diabetes (T1D) onset, with the most prominent changes involving sialylated glycans, hypothesized to mainly come from the Fab region, however, the analytical method used could not distinguish between Fc and Fab. Methods: IgG was isolated from plasma from 118 children with T1D and 98 healthy controls from the Danish Registry of Childhood and Adolescent Diabetes. Isolated IgG was cleaved into Fc and Fab fragments using IdeS enzyme. N-glycans were enzymatically released from each fragment, fluorescently labelled with procainamide, and analyzed separately using the UPLC-MS method. Structural annotation of resulting chromatograms was performed using MS/MS. Results: T1D related N-glycosylation changes were more pronounced in the Fab glycans compared to Fc glycans, with five Fab glycans (Man5, Man7, FA2BG1S1, A2G2S2, FA2BG2S1) being significantly altered compared to only one in the Fc region (FA2[3]BG1). Comparing Fc and Fab glycosylation overall reveals stark differences in the types of glycans on each region, with a more diverse and complex repertoire being present in the Fab region. Conclusions: These findings suggest that N-glycosylation changes in early onset T1D predominantly originate from the Fab region, underscoring their potential role in modulating (auto)immunity and highlighting distinct glycosylation patterns between Fc and Fab. Full article

The emergence of coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has become a global issue since 2019. The prominent characteristic of SARS-CoV-2 is the presence of the spike (S) protein protruding from the virus particle envelope. The S protein is a major drug and vaccine target because it initiates the key step in infection. Medicinal herbs are a potential treatment option to enhance immunity to fight viral infections. Caesalpinia sappan L. has been reported to display promising anti-viral activities. Specifically, brazilin (BRA), a major bioactive compound in C. sappan, was reported to play a role in inhibiting viral infection. Thus, the ability of BRA as a COVID-19 treatment was tested. The S protein was used as the BRA target of this work. Understanding the binding mechanism of BRA to the S protein is crucial for future utilisation of C. sappan as a COVID-19 treatment or other coronavirus-caused pandemics. Here, we performed molecular docking of BRA onto the S protein receptor binding domain (RBD) and multimerisation (MM) pockets. Molecular dynamics (MD) simulations were conducted to study the stability of binding to glycosylated and non-glycosylated S protein constructs. BRA can bind to the Receptor-binding motif (RBM) on an RBD surface stably; however, it is too large to fit into the MM pocket, resulting in dissociation. Nonetheless, BRA is bound by residues near the S1/S2 interface. We found that glycosylation has no effect on BRA binding, as the proposed binding site is far from any glycans. Our results thus indicate that C. sappan may act as a promising preventive and therapeutic alternative for COVID-19 treatment. Full article

Glycosaminoglycans (GAGs) are key ligands for proteins involved in physiological and pathological processes. Specific GAG-binding patterns are rarely identified, with the heparin pentasaccharide as an Antithrombin-III ligand being the best characterized. Generating glycan-specific antibodies is difficult due to their size, pattern dispersion, and flexibility. Single-domain variable new antigen receptors (VNAR nanobodies) from nurse sharks are highly soluble, stable, and versatile. Their unique properties suggest advantages over conventional antibodies, particularly for challenging biotherapeutic targets. Here we have used VNAR semi-synthetic phage libraries to select high-affinity fondaparinux-binding VNARs that did not show cross-reactivity with other GAG species. Competition ELISA and surface plasmon resonance identified a single fondaparinux-selective VNAR clone. This VNAR exhibited an extraordinarily stable protein fold: the beta-strands are stabilized by a robust hydrophobic network, as revealed by heteronuclear NMR. Docking fondaparinux to the VNAR structure revealed a large contact surface area between the CDR3 loop of the antibody and the glycan. Fusing the VNAR with a human Fc domain resulted in a stable product with a high affinity for fondaparinux (Kd = 9.3 × 10⁻⁸ M) that could efficiently discriminate between fondaparinux and other glycosaminoglycans. This novel glycan-targeting screening technology represents a promising therapeutic strategy for addressing GAG-related diseases. Full article

Background/Objectives: We aim to determine if cytokeratin-18 fragment (CK-18F) could be used to diagnose metabolic dysfunction-associated steatohepatitis (MASH). Methods: A total of 289 patients with metabolic dysfunction-associated steatotic liver disease (MASLD) were enrolled in the analysis. To evaluate the association between CK-18F levels and the histological features of MASH, weighted receiver operating characteristic (ROC) curve analyses were performed. The diagnostic utility of CK-18F was compared with that of the Mac-2 binding protein glycan isomer (M2BPGi). Additionally, we assessed the predictive performance of combining CK-18F with either the FIB-4 index or the FIB-3 index for diagnosing MASH and investigated predictors of future progression to cirrhosis. Results: CK-18F was more useful for MASH diagnosis than M2BPGi and the FIB-4 index in the multivariate analysis, with a sensitivity of 47% and specificity of 80% at a CK-18F cutoff value of 750 U/L. Because CK-18F decreases with advanced liver fibrosis, the combination of the FIB-4 or FIB-3 index with CK-18F was examined to identify cases with cirrhosis. The combination of the CK-18F level and the FIB-3 index better predicted MASH than the combination of the CK-18F level and the FIB-4 index. The FIB-3 index was the most useful predictor of cirrhosis on imaging five years after diagnosis with F2 or less disease. Conclusions: CK-18F is useful for MASH diagnosis, and the diagnostic algorithm combining CK-18F with the FIB-3 index may be more useful than the previously reported MASH diagnostic algorithm that combined it with the FIB-4 index. Full article