Search Results (281)

GNE myopathy is a rare genetic neuromuscular disorder caused by mutations in the GNE gene. The respective gene product, UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE), is a bifunctional enzyme that initiates endogenous sialic acid biosynthesis. Sialic acids are important building blocks for the glycosylation machinery of cells and are typically found at the terminal ends of glycoprotein N- and O-glycans. The exact pathomechanism of GNE myopathy remains elusive, and a better understanding of the disease is urgently needed for the development of therapeutic strategies. The purpose of this study was to examine the effects of hyposialylation on glycan structures and subsequent downstream effects in the C2C12 Gne knockout cell model. No overall remodeling of N-glycans was observed in the absence of Gne, but differences in glycosaminoglycan expression and O-GlcNAcylation were detected. Expression analysis of myopathogenes revealed concomitant down-regulation of muscle-specific genes. Among the top candidates were the sodium channel protein type 4 subunit α (Scn4a), voltage-dependent L-type calcium channel subunit α-1s (Cacna1s), ryanodine receptor 1 (Ryr1), and glycogen phosphorylase (Pygm), which are associated with excitation-contraction coupling and energy metabolism. The results suggest that remodeling of the glycome could have detrimental effects on intracellular signaling, excitability of skeletal muscle tissue, and glucose metabolism. Full article

The balance of alveolar macrophage (AM) polarization is severely disrupted in chronic inflammatory diseases like bronchiectasis, where a persistent pro-inflammatory (M1) phenotype perpetuates inflammation. To address this, we developed a high-throughput platform using a series of synthetic glycoligands (L1-L5) on a polyethyleneimine (PEI) scaffold. These ligands, which have varying affinities for macrophage lectin-like receptors, were used for phenotypic “fingerprinting” of AM subpopulations from pediatric bronchiectasis patients and a healthy control. Analysis of bronchoalveolar lavage fluid (BALF) revealed a pathogenic, M1-dominant profile (55% M1) in patients, confirming a state of chronic inflammation, which starkly contrasted with the quiescent, M0-dominant profile in the healthy control. We then leveraged this platform for targeted immunomodulation, using a drug-ligand conjugate to steer the dysregulated macrophage population toward a healthy state. The most potent conjugate, Dox-L5, dramatically suppressed the pathogenic M1 population (from 55% to 16%). This M1 suppression was accompanied by a significant shift toward the M2a (tissue-repair) phenotype and the emergence of a quiescent M0-like population, effectively remodeling the AM profile. This work validates a glycan-based platform for both diagnosing and correcting pathological macrophage imbalances. Our targeted approach offers a precise strategy to resolve chronic inflammation in bronchiectasis by suppressing M1 macrophages and promoting a pro-resolving M0/M2 phenotype, thereby restoring lung homeostasis. Full article

Clostridial neurotoxins, botulinum neurotoxins (BoNTs), and tetanus neurotoxin (TeNT) are potent toxins responsible for severe diseases, botulism and tetanus, respectively. BoNTs associate with non-toxic proteins (non-toxic non-hemagglutinin, hemagglutinins, and OrfXs), which protect BoNTs against acidic pH and protease degradation and facilitate BoNT passage through the intestinal barrier. TeNT enters motor neurons and undergoes a retrograde axonal transport until the target inhibitory interneurons in the central nervous system. BoNTs and TeNT recognize specific cell surface receptors which consist of complex sets of protein(s)-glycan-gangliosides and determine specific cell entry pathways. Recent data on structural and functional investigations of BoNT and TeNT receptors bring a better understanding of toxin trafficking in the host and entry into target neuronal cells, which is useful for the development of updated strategies of prevention and treatment of the corresponding diseases. Since clostridial neurotoxins, notably BoNTs, are important therapeutic tools, detailed knowledge of their activity opens the way of the development of engineered molecules for specific clinical applications. Full article

The COVID-19 pandemic accelerated vaccine innovation but also exposed weaknesses in global access and manufacturing. Yeast-based platforms, particularly Saccharomyces cerevisiae and Pichia pastoris, also known as Komagataella phaffii, offer a practical complement to vector systems. These eukaryotic microorganisms combine safety, scalability, and cost-effectiveness with the ability to express complex antigens and assemble virus-like particles. Building on the success of the recombinant hepatitis B vaccine, recent advances in glycoengineering, CRISPR-based host optimization, and surface display technologies have expanded the utility of yeast-based platforms for the rapid development of vaccines. Yeast-derived SARS-CoV-2 receptor-binding domain (RBD) subunit vaccines, such as Corbevax and Abdala (CIGB-66), demonstrate that affordable, immunogenic, and thermostable products are feasible at scale. Emerging innovations in glycan humanization, thermostable formulations, and oral or mucosal delivery highlight the potential of yeast-based vaccines for decentralized manufacturing and equitable pandemic preparedness. This review summarizes recent technical and clinical progress in yeast-based vaccine research, positioning these platforms as accessible and adaptable tools for future outbreak responses and global immunization strategies. 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

Human milk oligosaccharides (HMOs) are the third most abundant solid component in human milk and play crucial roles in shaping the gut microbiome and promoting infant health. Although their functions during infancy are well established, emerging evidence suggests that HMOs exert region-specific effects throughout the gastrointestinal tract, extending their benefits beyond early life. This review summarizes current findings on HMO activity in the oral cavity, stomach, small intestine, and large intestine, focusing on their microbiota-modulating, barrier-enhancing, and immunoregulatory effects. In the oral cavity, HMOs inhibit pathogen adhesion and biofilm formation, maintaining oral homeostasis. In the stomach, fucosylated and sialylated HMOs act as soluble decoy receptors, preventing Helicobacter pylori infection. In the small intestine, HMOs strengthen epithelial integrity, regulate inflammation, and promote nutrient absorption. In the large intestine, they serve as selective prebiotics for beneficial microbes, enhancing short-chain fatty acid production and improving barrier function. Although preclinical and clinical studies demonstrate their safety and efficacy, further research is required to elucidate their mechanisms in adults. Overall, HMOs represent multifunctional bioactive glycans with promising applications for gastrointestinal health across all ages. 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

The vomeronasal system (VNS) is directly linked to the various behavior and ecology of all animal species, and understanding it might help to prevent deer damage. We therefore histochemically analyzed the accessory olfactory bulb (AOB) and the vomeronasal organ (VNO) that, respectively, function as a primary integrative center and a peripheral receptor organ, in Hokkaido sika deer (Cervus nippon ssp. yesoensis). The AOB consisted of the vomeronasal nerve, glomerular, plexiform, and granule cell layers. We found that G protein α subunit i2 (Gαi2) and o (Gαo) that are, respectively, coupled with vomeronasal receptor type 1 and 2 families were strongly and weakly expressed in the glomerular layer, respectively. These properties of the AOB of sika deer were similar to those of other artiodactyl species, including wapiti. We then explored the sika deer VNO using 21 lectins that bind to each glycan structure. Although various cell types in the VNO had unique lectin binding profiles, all 21 lectins bound to the free border of the sensory epithelium, suggesting that various glycoconjugates are involved in pheromone detection in sika deer via the VNO. Furthermore, the reactivity of some lectins in the sensory epithelium and vomeronasal gland differed from those of roe deer and wapiti. Our findings suggest that the composition of glycoconjugates in the VNO differs among deer species. Full article

Alzheimer’s disease (AD) is the most prevalent cause of dementia, characterized by progressive cognitive decline, amyloid-β (Aβ) plaques, and neurofibrillary tangles composed of hyperphosphorylated tau protein. Other tauopathies, including frontotemporal lobar degeneration (FTLD), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD) share pathological hallmarks centered on abnormal tau biology. Increasing evidence highlights the role of post-translational modifications in modulating these pathogenic processes. Among these, glycosylation, the enzymatic attachment of glycans to proteins or lipids, has emerged as a critical regulator of protein folding, trafficking, aggregation, and clearance. Both N-linked glycosylation (N-glycosylation) and O-linked glycosylation (O-glycosylation) influence tau stability, Aβ processing, receptor signaling, synaptic integrity, and neuroinflammation. Dysregulated glycosylation patterns have been documented in brains and cerebrospinal fluid (CSF) of AD patients, suggesting biomarker potential and novel therapeutic targets. Moreover, glycosyltransferases and glycosidases show altered expression in neurodegeneration, linking metabolic and inflammatory pathways to tauopathy progression. This review synthesizes current evidence on the implications and consequences of glycosylation in AD and other tauopathies, integrating mechanistic, pathological, and clinical findings. We also discuss advances in glycoproteomics, the interplay between glycosylation and phosphorylation, and the translational potential of targeting glycosylation pathways for diagnosis and therapy. Full article

The H3N8 low pathogenic avian influenza virus (LPAIV) exhibits broad host tropism, infecting diverse avian and mammalian species, raising concerns about its zoonotic potential. Following the emergence of human infections with H3N8 LPAIV in China, including a fatal case, we investigated the epidemiological and virological characteristics of this virus in Guangdong Province. In 2022, a serological survey revealed H3N8 seroprevalence rates of 10.85% in farmed chickens and 7.97% in ducks. We isolated three H3N8 viruses, designated as A/chicken/Qingyuan/22/2022 (H3N8); A/chicken/Qingyuan/31/2022 (H3N8); and A/chicken/Qingyuan/15/2022 (H3N8), and found that these chicken isolates, like the human isolate A/Changsha/1000/2022, share the same E190 residue. This residue can synergize with sites such as Q226 and G228 to enhance binding affinity for SAα-2,6-Gal. Additionally, they harbor the three amino acid residues N193, W222, and S227. Among these, N193 has the potential to form hydrogen bonds with α2-6-linked glycans, while W222 and S227 may alter the conformational flexibility of the 220-loop. These two effects collectively endow the H3N8 isolates with dual receptor-binding properties. These findings suggest a shift in receptor specificity, potentially facilitating viral adaptation to mammalian hosts. Characterization of viral genome detection dynamics, and histopathology in animal models further elucidated the viral infection dynamics. Our study provides critical insights into the evolutionary trajectory and zoonotic potential of the H3N8 LPAIV. Full article

Vaccine adjuvants play a crucial role in the field of vaccinology, yet they remain one of the least developed and poorly characterized components of modern biomedical research. The limited availability of clinically approved adjuvants highlights the urgent need for new molecules with well-defined mechanisms and improved safety profiles. Hemocyanins, large copper-containing metalloglycoproteins found in mollusks, represent a unique class of natural immunomodulators. Hemocyanins serve as carrier proteins that help generate antibodies against peptides and hapten molecules. They also function as non-specific protein-based adjuvants (PBAs) in both experimental human and veterinary vaccines. Their mannose-rich N-glycans allow for multivalent binding to innate immune receptors, including C-type lectin receptors (e.g., MR, DC-SIGN) and Toll-like receptor 4 (TLR4), thereby activating both MyD88- and TRIF-dependent signaling pathways. Hemocyanins consistently favor Th1-skewed immune responses, which is a key characteristic of their adjuvant potential. Remarkably, their conformational stability supports slow intracellular degradation and facilitates dual routing through MHC-II and MHC-I pathways, thereby enhancing both CD4+ and CD8+ T-cell responses. Several hemocyanins are currently being utilized in biomedical research, including Keyhole limpet hemocyanin (KLH) from Megathura crenulata, along with those from other gastropods such as Concholepas concholepas (CCH), Fissurella latimarginata (FLH), Rapana venosa (RvH), and Helix pomatia (HpH), all of which display strong immunomodulatory properties, making them promising candidates as adjuvants for next-generation vaccines against infectious diseases and therapeutic immunotherapies for cancer. However, their structural complexity has posed challenges for their recombinant production, thus limiting their availability from natural sources. This reliance introduces variability, scalability issues, and challenges related to regulatory compliance. Future research should focus on defining the hemocyanin immunopeptidome and isolating minimal peptides that retain their adjuvant activity. Harnessing advances in structural biology, immunology, and machine learning will be critical in transforming hemocyanins into safe, reproducible, and versatile immunomodulators. This review highlights recent progress in understanding how hemocyanins modulate mammalian immunity through their unique structural features and highlights their potential implications as potent PBAs for vaccine development and other biomedical applications. By addressing the urgent need for novel immunostimulatory platforms, hemocyanins could significantly advance vaccine design and immunotherapy approaches. Full article

The glycan profile of cells comprises a high variety of sugar moieties that are attached to proteins (glycoproteins) and lipids (glycolipids) via a process called ‘glycosylation’. Cancer cells commonly carry aberrant glycans, which may be of interest for cancer diagnosis, prognosis, as well as the development of novel therapeutic strategies. This review focuses on the differential glycosylation patterns on malignant B cells, including both B cell lymphoma and leukemia. Well-known aberrant glycan profiles on malignant B cells include acquired high mannose N-glycans in the B cell receptor (BCR) of follicular lymphoma (FL), and increased expression of the glycosphingolipid Gb3/CD77 on Burkitt’s lymphoma (BL). These structures can be exploited for therapy by using lectins that specifically recognize these patterns with intrinsic cytotoxic activity or in a drug-conjugate format. Furthermore, immunotherapy can be improved by modulating glycans, especially sialic acids. Targeting glycans for immunotherapy is also of interest for chimeric antigen receptor (CAR) T cell therapy, a relatively novel therapy that has been quite effective in various B cell malignancies. Thus, the glycan profile of malignant B cells harbors many opportunities for therapeutic targeting. It is anticipated that further in-depth glycan profiling will open up many more opportunities for the treatment of B cell malignancies. Full article

Background: Antibody-dependent cellular cytotoxicity (ADCC) is an immune response where antibodies bind to target cells and activate effector cells through Fcγ receptors, ultimately leading to the destruction of the target cells. Methods: This study examined the ADCC activities of charge variants of a therapeutic IgG₁, MAB1, using an internally developed reporter gene assay. In this assay, the proprietary target was expressed on DiFi cells, while FcγRIIIa was expressed on Jurkat effector cells. Results: The results revealed that different charge variants had varying levels of ADCC activity, with variants containing C-terminal lysine residues showing enhanced activity. The charge variants arose from modifications such as the presence of sialic acid at the glycan moiety, deamidation, and C-terminal lysine truncation, including K2 (two C-terminal lysine residues), K1 (one C-terminal lysine residue), and K0 (no C-terminal lysine residues) variants. Notably, the K1 and K2 variants demonstrated higher ADCC activity compared to the K0 and acidic variants. However, the observed increase was attributed not to the lysine residue itself, but rather to the MAN5 glycan associated with the lysine-containing variants. Conclusion: These findings challenge previous assumptions about the role of C-terminal lysine in ADCC, suggesting a shift in understanding the functional significance of charge variants and emphasizing the critical influence of glycan composition in therapeutic antibody efficacy. Full article

Antibodies are a cornerstone of the adaptive immune response, serving as key defenders against viral infections; however, they can also act as a double-edged sword, contributing to immune-mediated pathologies. This review advances a “Yin-Yang” framework to integrate the dual activities of antibodies. The protective ‘Yin’ functions are driven by high-affinity antibodies generated through processes like somatic hypermutation and class-switch recombination. These antibodies execute viral neutralization, activate the complement system, and engage Fc receptors (FcRs) to drive antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis. These mechanisms form the immunological basis of effective vaccines, which aim to elicit durable and functionally specialized antibody isotypes like IgG and mucosal IgA. Conversely, the pathogenic ‘Yang’ of the response can be detrimental. This includes antibody-dependent enhancement (ADE) of infection, notably observed with flaviviruses, and the development of autoimmunity through mechanisms like molecular mimicry and bystander activation, which can lead to conditions such as multiple sclerosis and Guillain-Barré Syndrome. The balance between protection and pathology is tipped by a confluence of factors. These include viral evasion strategies like antigenic mutation and glycan shielding, as well as host-based determinants such as genetic polymorphisms in FcRs, immune history, and the gut microbiome. Understanding these molecular determinants informs the rational design of next-generation interventions. Promising strategies, such as Fc-region glyco-engineering and the design of tolerogenic vaccines, aim to selectively promote protective functions while minimizing pathological risks, offering a clear path forward in combating viral threats. Full article

Background/Objectives: This study comprehensively characterized the O- and N-glycosylation profiles of bispecific antibodies (BsAbs) via advanced analytical techniques to evaluate their structural and functional implications. Methods: High-resolution MS revealed O-xylosylation at Ser468 within the (G4S)4 linker peptide, which was identified as xylose with a molecular weight of 132.042 Da. HILIC-HPLC analysis of N-glycosylation revealed glycan species engineered to eliminate Fc effector functions. O-glycosylation analysis via β-elimination followed by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) identified xylose as the predominant glycan. Results: O-xylosylation does not affect the binding of BsAbs to either antigen Programmed Death-1 (PD-1) or Vascular Endothelial Growth Factor (VEGF). Notably, O-xylosylation interactions with mannose receptor represent the first discovery highlighting potential immunomodulatory roles. Conclusions: This study highlights the critical importance of monitoring comprehensive glycosylation characterization during the development of BsAb with (G4S)n linkers to ensure optimal therapeutic efficacy, safety, and reduced immunogenic potential. Full article