Search Results (164)

Background: Human noroviruses are the leading cause of foodborne gastroenteritis worldwide. Accumulating evidence suggests that food matrices containing fucosylated or histo-blood group antigen (HBGA)-like glycans may facilitate viral attachment and persistence, yet the molecular mechanisms underlying these interactions remain unclear. Methods: In this study, we performed a comparative computational analysis of norovirus–glycan interactions by integrating AlphaFold3-based structure prediction, molecular docking, and molecular dynamics simulations. A total of 182 P-domain models representing all genotypes across five human norovirus genogroups (GI, GII, GIV, GVIII, and GIX) were predicted and docked with a lettuce-derived core-fucosylated hexasaccharide (H₂N₂F₂) previously identified by our group. The three complexes exhibiting the most favorable docking energies were further examined using 40 ns molecular dynamics simulations, followed by MM/GBSA binding free energy calculations and per-residue decomposition analyses. Results: Docking results indicated that the majority of modeled P proteins were able to adopt energetically favorable interaction poses with H₂N₂F₂, with predicted binding energies ranging from −3.7 to −7.2 kcal·mol⁻¹. The most favorable docking energies were observed for GII.6_S9c_KC576910 (−7.2 kcal·mol⁻¹), GII.3_MX_U22498 (−7.1 kcal·mol⁻¹), and GII.4_CARGDS11182_OR700741 (−6.8 kcal·mol⁻¹). Molecular dynamics simulations suggested stable ligand engagement within canonical HBGA-binding pockets, with recurrent residues such as Asp374, Gln393, and Arg345 contributing to electrostatic and hydrophobic interactions, consistent with previously reported HBGA-binding motifs. MM/GBSA analyses revealed comparatively favorable binding tendencies among these complexes, particularly for globally prevalent genotypes including GII.3, GII.4, and GII.6. Conclusions: This work provides a large-scale structural and energetic assessment of the potential interactions between a naturally occurring lettuce-derived fucosylated hexasaccharide and human norovirus P domains. The results support the notion that core-fucosylated food-associated glycans can serve as interaction partners for diverse norovirus genotypes and offer comparative molecular insights into glycan recognition patterns relevant to foodborne transmission. The integrative AlphaFold3–docking–dynamics framework presented here may facilitate future investigations of virus–glycan interactions within food matrices. Full article

A key function of naturally occurring antibodies is to control pathologically altered cells, such as those with aberrant glycosylation. Age-related diminution in the pool of B cells producing these immunoglobulins is linked to impaired anti-tumor immunity. In this study, the levels of antibodies against tumor-associated carbohydrate antigens (TACAs)—common in gastric cancer (GC) and other malignancies—were analyzed in 235 treatment-naïve GC patients (stages I–IV) and 76 healthy donors using a printed glycan array (PGA). We found that anti-glycan IgM levels, but not IgG, reduced with age in both patients and donors. Crucially, IgM levels against most glycans were significantly lower in the GC cohort compared with healthy donors, a trend that remained after age adjustment. Furthermore, an immunohistochemical analysis revealed that human anti-GalNAcα (Tn) antibodies—a well-characterized TACA in gastrointestinal cancers—bound to tumor cells and exhibited perinuclear and membrane staining in non-tumor surface cells within the same organ. These data support the hypothesis that gastric cancer patients have reduced levels of anti-glycan IgMs, which are responsible for the early recognition of transformed cells. This specific immunodeficiency may contribute to a permissive environment for tumor development. Full article

C-type lectins (CTLs) are a large family of calcium-dependent carbohydrate-binding proteins that play crucial roles in innate immunity as pattern recognition receptors. Bivalve mollusks possess exceptionally diverse and expanded repertoires of CTLs, yet a systematic review integrating their structural, functional, and regulatory aspects has been lacking. This article provides a comprehensive synthesis of current knowledge on bivalve CTLs, analyzing their biosynthesis, complex tissue-specific expression under both normal and stressed conditions, and their multifaceted roles in immune defense and other physiological processes. Our analysis consolidates data on their diverse domain architectures, phylogenetic relationships, and the variability of key motifs within their carbohydrate-recognition domains. The results demonstrate that bivalve CTLs are not only critical for pathogen recognition, agglutination, and phagocytosis but also involved in processes like nutrition, development, byssus formation and biomineralization. However, a significant finding is that the detailed carbohydrate specificity for most bivalve CTLs remains poorly characterized, often limited to monosaccharide inhibition assays. In conclusion, while the immune role of bivalve CTLs is well-established, this review underscores a critical gap in understanding their fine glycan-binding profiles. Therefore, a shift in the focus of future research towards elucidating their structure and carbohydrate specificity is required for a full understanding of their biological functions and an assessment of their biomedical potential. Full article

Novel adjuvants are key to making allergen-specific immunotherapy (AIT) safer and more effective. Their development is crucial for moving AIT into a new generation of precision medicine. N-glycosylation of protein antigens plays a pivotal role in modulating innate immune responses through enhanced recognition by pattern recognition receptors. New AIT vaccine strategies aim to exploit this by using innate-targeting adjuvants, modifying allergen structures, and routing early responses toward tolerance. Thus, we engineered five distinct N-glycosylated adjuvant configurations, composed of the receptor-binding domain of hemagglutinin (H1s) and Der p 2 (D2) allergen, to explore how glycan profile affects innate immune response for the application in therapeutic strategies for Type 1 hypersensitivity. Glycoengineered proteoforms produced in Pichia pastoris were structurally verified by mass spectrometry. Using M0 and M2 THP-1-derived macrophages, binding of all H1sD2 proteoforms to DC-SIGN was confirmed via confocal microscopy and flow cytometry. Stimulation of PBMCs with these proteoforms led to increased IL-10 and IFN-γ levels, indicating a shift toward regulatory immune responses. Notably, the M2 glycovariant elicited the strongest immunomodulatory signature, suggesting significant promise as a therapeutic candidate. These findings support the potential of glycoengineered allergen-adjuvant proteoforms to fine-tune innate immunity and improve the safety and efficacy of AIT. Full article

Galectins, a family of glycan-binding proteins, play crucial roles in various cellular functions, acting at both intracellular and extracellular levels. Among them, Galectin-3 (Gal-3) stands out as a unique member, possessing an intrinsically unstructured N-terminal oligomerization domain and a canonical carbohydrate-recognition domain (CRD). Gal-3 binding to glycosylated plasma membrane cargo leads to its oligomerization and membrane bending, ultimately resulting in the formation of endocytic invaginations. An interactomic assay using proteomic analysis of endogenous Gal-3 immunoprecipitates identified the orphan G protein-coupled receptor GPRC5A as a novel binding partner of Gal-3. GPRC5A, also known as Retinoic Acid-Induced protein 3 (RAI3), is transcriptionally induced by retinoic acid. Our results further demonstrate that extracellular recombinant Gal-3 stimulates GPRC5A internalization. In SW480 colorectal cancer cells, glycosylated GPRC5A interacts with Gal-3. Interestingly, while GPRC5A expression was upregulated by the addition of all-trans retinoic acid (ATRA), its endogenous internalization in SW480 cells was specifically triggered by extracellular Gal-3, but not by ATRA. This study provides new insights into the endocytic mechanisms of GPRC5A, for which no specific ligand has been identified to date. Further research may uncover additional Gal-3-mediated functions in GPRC5A cellular signaling and contribute to the development of innovative therapeutic strategies. Full article

Sialyl-Tn (sTn) is a tumor-associated carbohydrate antigen (TACA) abundantly expressed by various types of carcinomas. While conventional antibody-based platforms have traditionally been used for the detection and targeting of sTn, alternative binding scaffolds may offer distinct advantages. Variable lymphocyte receptor B (VLRB), the immunoglobulin-like molecule of jawless vertebrates, offers a promising alternative for glycan recognition. In this study, a phage-displayed VLRB library was utilized to identify sTn-specific binders. Two candidates, designated as ccombodies A8 and B11, were isolated after four rounds of biopanning. Both were expressed and purified using Ni-affinity and FPLC, yielding proteins with apparent molecular weights of ~27 kDa in SDS-PAGE. Sequence analysis revealed a preference for glycan-binding residues in randomized hypervariable regions, with A8 exhibiting an increased aliphatic content. ELISA confirmed selective binding to sTn and other O-glycans containing the core α-GalNAc, with EC₅₀ values of 18.2 and 14.2 nM for A8 and B11, respectively. Vicia villosa lectin inhibited ccombody binding to sTn, indicating shared epitope recognition. Additionally, both ccombodies bound to sTn-positive glycoproteins and carcinoma cell lines HeLa and LS174T. These findings demonstrate that phage display of VLRBs enables the identification of high-affinity, glycan-specific binders, offering a compelling alternative to immunoglobulin-based platforms for future diagnostic and therapeutic applications targeting tumor-associated glycans. Full article

Within recent years, the interest in tools to investigate carbohydrate-protein (CPI) and carbohydrate-carbohydrate interactions (CCI) has increased significantly. For the investigation of CPI and CCI, several techniques employing different linking methods are available. For mimicking the glycocalyx self-assembled monolayer (SAM) formation of carbohydrate derivatives on gold nanoparticles is most appropriate. In contrast to methods for glyco-SAM formation used previously to analyze CPI/CCI, the novel approach allows for a facile and rapid synthesis to link spacers and carbohydrate derivatives and enhances the binding event by controlling the amount and orientation of ligand. For immobilization on biorepulsive aminooxy functionalized gold nanoparticles by oxime coupling, diverse aldehyde-functionalized glycan structures of mono-, di-, and complex trisaccharides were synthesized, employing several facile steps including olefin metathesis. Effective immobilization and first binding studies are presented for the lectin concanavalin A. This novel and advantageous immobilization method is presently employed in various biomimetic studies of carbohydrates and carbohydrate-based array development for diagnostics and screening. Full article

The interactions of leukocyte glycoproteins with adhesion and signaling molecules through glycan recognition are not well understood. We previously demonstrated that galectin-8, a tandem-repeat lectin with N- and C-terminal carbohydrate binding domains which is highly expressed in endothelial and epithelial cells, can bind to activated neutrophils to induce surface exposure of phosphatidylserine (PS) without DNA fragmentation or apoptosis, in a process termed preaparesis. However, the receptors for Gal-8 on leukocytes have not been identified. Here we report our results using both proteomics and affinity chromatography with both full-length Gal-8 and the separate Gal-8 C-terminal and N-terminal domains to identify glycoprotein ligands in HL-60 cells for Gal-8. Two of the major ligands for Gal-8 are CD45RA and CD45RC (Protein Tyrosine Phosphatase, PTP) and basigin (CD147). Both CD45 and basigin are integral membrane glycoproteins that carry poly-N-acetyllactosamine modifications on N- and/or O-glycans, required for Gal-8 binding. Inhibition of the phosphatase activity of CD45 reduced Gal-8-induced PS exposure, indicating a possible role of CD45 in Gal-8 signaling of preaparesis in human leukocytes. These results demonstrate unique glycoprotein recognition by Gal-8 involved in cell recognition and signaling. Full article

Alzheimer’s disease (AD), a progressive neurodegenerative disorder, is marked by the pathological accumulation of amyloid-β plaques and tau neurofibrillary tangles, both of which disrupt neuronal communication and function. Emerging evidence highlights the role of extracellular vesicles (EVs) as key mediators of intercellular communication, particularly in the propagation of pathological proteins in AD. Among the regulatory factors influencing EV composition and function, neuraminidase 1 (NEU1), a lysosomal sialidase responsible for desialylating glycoproteins has gained attention for its involvement in EV glycosylation. This review explores the role of NEU1 in modulating EV glycosylation, with particular emphasis on its influence on immune modulation and intracellular trafficking pathways and the subsequent impact on intercellular signaling and neurodegenerative progression. Altered NEU1 activity has been associated with abnormal glycan profiles on EVs, which may facilitate the enhanced spread of amyloid-β and tau proteins across neural networks. By regulating glycosylation, NEU1 influences EV stability, targeting and uptake by recipient cells, primarily through the desialylation of surface glycoproteins and glycolipids, which alters the EV charge, recognition and receptor-mediated interactions. Targeting NEU1 offers a promising therapeutic avenue to restore EV homeostasis and reduces pathological protein dissemination. However, challenges persist in developing selective NEU1 inhibitors and effective delivery methods to the brain. Furthermore, altered EV glycosylation patterns may serve as potential biomarkers for early AD diagnosis and monitoring. Overall, this review highlights the importance of NEU1 in AD pathogenesis and advocates for deeper investigation into its regulatory functions, with the aim of advancing therapeutic strategies and biomarker development for AD and related neurological disabilities. 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

Alzheimer’s disease (AD) is one of the most common neurodegenerative disorders linked to aging. Major hallmarks of AD pathogenesis include amyloid-β peptide (Aβ) plaques, which are extracellular deposits originating from the processing of the amyloid precursor protein (APP), and neurofibrillary tangles (NFTs), which are intracellular aggregates of tau protein. Recent evidence indicates that disruptions in metal homeostasis and impaired immune recognition of these aggregates trigger neuroinflammation, ultimately driving disease progression. Therefore, a more comprehensive approach is needed to understand the underlying causes of the disease. Patients with AD present abnormal glycan profiles, and most known AD-related molecules are either modified with glycans or involved in glycan regulation. A deeper understanding of how O-glycosylation influences the balance between amyloid-beta peptide production and clearance, as well as microglia’s pro- and anti-inflammatory responses, is crucial for deciphering the early pathogenic events of AD. This review aims to provide a comprehensive summary of the extensive research conducted on the role of mucin-type O-glycosylation in the pathogenesis of AD, discussing its role in disease onset and immune recognition. Full article

The growing recognition of the role of milk-derived exosomes in metabolic and immunological processes has brought attention to the potential utility of donkey milk. However, the efficacy and bioactive components of donkey milk are underexplored. This study aimed to elucidate the proteomic profiles of exosomes isolated from donkey colostrum and mature milk using advanced four-dimensional (4D) label-free quantitative proteomics. A comprehensive analysis identified and quantified a total of 2293 exosomal proteins from donkey milk, including 276 differentially expressed exosomal proteins (DEEPs). The results revealed marked proteomic differences between colostrum and mature milk exosomes, particularly in proteins associated with immune responses and metabolic pathways. Exosomal proteins derived from colostrum were found to be enriched in immune-modulatory factors and glycan-related pathways, which may contribute to the enhancement in neonatal immune system development. In contrast, exosomal proteins from mature milk were predominantly associated with metabolic processes and cellular senescence. Protein–protein interaction (PPI) analysis further suggested that specific exosomal proteins highly expressed in colostrum could serve as nutraceutical components with potential health benefits for humans. In conclusion, this study underscores the distinct proteomic features and potential physiological roles of exosomes from donkey colostrum versus mature milk. Full article

For the improvement of biosensor performance, the development of a molecular recognition material as well as a sensor platform is necessary. A glycopolymer is a molecular recognition material capable of recognizing specific proteins as natural glycans. However, the target molecules for biosensors using glycopolymers are limited to lectins that are already known for their specific interactions with glycan residues. The aim of this study is to investigate a glycopolymer-modified (GM) surface capable of recognizing non-lectin proteins. As non-lectin proteins, we focused on cytokines, in which the interaction preference to glycopolymers is unknown. The cytokine adsorption onto the GM surfaces was evaluated using a surface plasmon resonance imaging technique as a biosensing tool. Differences in cytokine adsorption onto the different glycan residues were revealed, which will be important for selective cytokine detection. This study indicates the possibility of a biosensing surface modified with glycopolymers for the detection of non-lectin proteins. The results are beneficial for expanding the use of glycopolymers as a molecular recognition material for future applications such as cell analysis and diagnostic devices. Full article

Background/Objectives: Abnormal glycan formation on the cancer cell surface plays a crucial role in regulating tumor functions in bladder cancer. In this study, we investigated the roles of glucosaminyl (N-acetyl) transferase 2 (GCNT2) in bladder cancer progression and immune evasion. GCNT2 synthesizes I-branched polylactosamine chains on cell surface glycoproteins. Understanding its functions will provide insights into tumor–immune interactions, facilitating the development of effective immunotherapeutic strategies. Methods: GCNT2 expression levels in bladder cancer cell lines and patient tumor samples were analyzed via quantitative polymerase chain reaction and immunohistochemistry. GCNT2 functions were assessed via overexpression and knockdown experiments. Its effect on natural killer (NK) cell-mediated cytotoxicity was evaluated via in vitro assay. Cytotoxic granule release from NK cells was measured via enzyme-linked immunosorbent assay. Results: GCNT2 expression was inversely correlated with bladder cancer aggressiveness in both cell lines and patient samples. Low GCNT2 levels were associated with advanced tumor stage and grade, suggesting the tumor-suppressive roles of GCNT2. Notably, GCNT2 overexpression enhanced the susceptibility of bladder cancer cells to NK cell-mediated killing, whereas its knockdown promoted immune evasion. GCNT2-overexpressing cells strongly induced the release of cytotoxic granules from NK cells, indicating enhanced immune recognition. Conclusions: Our findings suggest that aggressive bladder tumors evade NK cell immunity by decreasing the GCNT2 levels and that I-antigen glycans synthesized by GCNT2 are crucial for NK cell recognition by tumor cells. Our findings provide insights into the tumor–immune interactions in bladder cancer and GCNT2 and its associated pathways as potential targets for novel immunotherapeutic strategies. Full article

Aging is often a choice between developing cancer or autoimmune disorders, often due in part to loss of self-tolerance or loss of immunological recognition of rogue-acting tumor cells. Self-tolerance and cell recognition by the immune system are processes very much dependent on the specific signatures of glycans and glycosylated factors present on the cell plasma membrane or in the stromal components of tissue. Glycosylated factors are generated in nearly innumerable variations in nature, allowing for the immensely diverse role of these factors in aging and flexibility necessary for cellular interactions in tissue functionality. In previous studies, we showed that differential expression of TMEM230, an endoplasmic reticulum (ER) protein was associated with specific signatures of enzymes regulating glycan synthesis and processing and glycosylation in rheumatoid arthritis synovial tissue using single-cell transcript sequencing. In this current study, we characterize the genes and pathways co-modulated in all cell types of the synovial tissue with the enzymes regulating glycan synthesis and processing, as well as glycosylation. Genes and biological and molecular pathways associated with hallmarks of aging were in mitochondria-dependent oxidative phosphorylation and reactive oxygen species synthesis, ER-dependent stress and unfolded protein response, DNA repair (UV response and P53 signaling pathways), and senescence, glycolysis and apoptosis regulation through PI3K-AKT-mTOR signaling have been shown to play important roles in aging or neurodegeneration (such as Parkinson’s and Alzheimer’s disease). We propose that the downregulation of TMEM230 and RNASET2 may represent a paradigm for the study of age-dependent autoimmune disorders due to their role in regulating glycosylation, unfolded protein response, and PI3K-AKT-mTOR signaling. Full article