Search Results (160)

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

Forest musk deer (Moschus berezovskii Flerov), a critically endangered ruminant species, faces extinction risks, with captive populations further threatened by prevalent digestive and immune disorders. This study utilized comparative metagenomic sequencing to assess intestinal microbiota structure and functional profiles between wild populations in Chongqing and Hunan and captive individuals. Wild populations exhibited a Pseudomonadota-dominated gut microbiota (significantly more abundant than in captive counterparts), enriched with lignin-degrading genera Novosphingobium and Acinetobacter. In contrast, the captive group demonstrated increased abundances of Bacillota/Bacteroidota, alongside abnormal proliferation of Escherichia and Clostridium. Both alpha and beta diversity analyses confirmed significant compositional divergences among the three groups, with wild populations maintaining higher diversity than captive populations. Notably, while substantial disparities in microbial abundance existed between wild populations (attributed to habitat vegetation differences), core microbial diversity and carbohydrate metabolic functions exhibited convergence. Functional analyses marked divergences in metabolic pathways: Captive microbiota showed enrichment in translation and glycan metabolism pathways, whereas wild populations displayed pronounced enrichment in immune regulation and environmental sensing pathways. These findings establish a theoretical foundation for optimizing wild population conservation strategies and developing science-based captive management protocols. 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

β-Lactosylceramide (β-LacCer) is not only a key intermediate in the biosynthesis of complex glycosphingolipids (GSLs) but also an important regulator of many biological processes. To facilitate the investigation of β-LacCer and other GSLs, a series of β-LacCer analogs with an azido group at the 6-C-position of the D-galactose in lactose and varied forms of the ceramide moiety were synthesized from commercially available lactose in sixteen linear steps by a versatile and diversity-oriented strategy, which engaged lipid remodeling and glycan functionalization at the final stage. These azide-labeled β-LacCer analogs are flexible and universal platforms that are suitable for further functionalization with other molecular tags via straightforward and biocompatible click chemistry, thereby paving the way for their application to various biological studies. Full article

We investigated HIV-1 immune evasion mechanisms in a slow progressor (CBJC515) by constructing pseudoviruses expressing autologous Env proteins. Intriguingly, all pseudoviruses exhibited resistance to the broadly neutralizing antibody (bNAb) PGT135. Using site-directed mutagenesis and chimeric Env construction, we identified distinct escape mechanisms: early 2005 strains lost the N332 glycan site, while 2006/2008 strains retained key epitopes but developed resistance through structural modifications in the V1/V4/C2 regions or acquired novel N-glycosylation sites (N398/N611). These findings provide insights into how HIV-1 can escape from N332-directed bNAb responses without altering the epitope itself. Furthermore, chimeric experiments also elucidated regional co-evolution and functional maintenance: the V1V2 region broadly interfered with envelope protein function, while the V3 region may exhibit compensatory activity, restoring functionality and mitigating deleterious polymorphisms in other regions to keep Env antigenic diversity. These results offer valuable mechanistic clues that may inform the development of next-generation HIV-1 vaccines. Full article

Breast cancer (BC) is a major global health concern, and early detection is key to improving patient outcomes. Aberrant glycosylation, particularly the sulfation of glycans, is implicated in cancer progression; however, analyzing these low-abundance glycans is challenging. This study aimed to profile serum sulfated N-glycans in Ethiopian patients with BC to identify novel biomarkers for the early detection of BC. Using a glycoblotting-based sulphoglycomics workflow, including high-throughput glycoblotting enrichment, weak anion exchange (WAX) separation, and MALDI-TOF MS, serum samples from 76 BC patients and 20 healthy controls were analyzed. Statistical evaluation revealed significant differences in the sulfated N-glycan profiles. Seven mono-sulfated N-glycans were markedly elevated in patients with BC, demonstrating high diagnostic accuracy (AUC ≥ 0.8) in this internal cohort. Terminal Lewis-type glycan epitopes were prominent in sulfated glycans but were absent in their non-sulfated counterparts. The increased fucosylation and sialylation of sulfated glycans are statistically significant markers of early-stage BC. The preservation of sialic acid groups during the analysis ensured detailed structural insight. This pioneering study quantitatively examined sulfated N-glycans in BC and identified potential glyco-biomarkers for early detection. Validation in larger, diverse cohorts is needed to establish their broader diagnostic relevance and improve our understanding of cancer-associated glycomic alterations. Full article

Inflammatory bowel disease (IBD), which includes Crohn’s Disease (CD) and Ulcerative Colitis (UC), is a chronic gastrointestinal (GI) disorder affecting 1 in 100 people in the United States. Pediatric IBD (PIBD) is estimated to impact 15 per 100,000 children in North America. Factors such as the gut microbiome (GM), genetic predisposition to the disease, and certain environmental factors are thought to be involved in pathogenesis. However, the pathophysiology of IBD is incompletely understood, and diagnostic biomarkers and effective treatments, particularly for PIBD, are limited. Recent work suggests that these factors may interact to influence disease development, and multiomic approaches have emerged as promising tools to elucidate the pathophysiology. We employed metagenomics, metabolomics- and metatranscriptomics-based approaches to examine the microbiome, its genetic potential, and its activity to identify factors associated with PIBD. Metagenomics-based analyses revealed pathways such as octane oxidation and glycolysis that were differentially expressed in UC patients. Additionally, metatranscriptomics-based analyses suggested enrichment of glycan degradation and two component systems in UC samples as well as protein processing in the endoplasmic reticulum, ribosome, and protein export in CD and UC samples. In addition, metabolomics-based approaches revealed patterns of differentially abundant metabolites between healthy and PIBD individuals. Interestingly, overall microbiome community composition (as measured by alpha and beta diversity indices) did not appear to be associated with PIBD. However, we observed a small number of differentially abundant taxa in UC versus healthy controls, including members of the Classes Gammaproteobacteria and Clostridia as well as members of the Family Rikenellaceae. Accordingly, when identifying potential biomarkers for PIBD, our results suggest that multiomics-based approaches afford enhanced potential to detect putative biomarkers for PIBD compared to microbiome community composition sequence data alone. Full article

The structural analysis of glycans remains a major challenge due to their high isomeric complexity and conformational flexibility arising from diverse glycosidic linkages and dynamic three-dimensional structures. Ion mobility–mass spectrometry (IM–MS) has been attracting attention as a way to develop the structural analysis of glycans. In this study, the effects of ionization conditions—including different types of adduct ions, ionization temperatures, and solvent environments—on the ion mobility behavior of glycans were systematically investigated. IM–MS measurements of ethylamine-tagged glycans showed broad arrival time distributions of monoprotonated ions indicating the presence of multiple conformers of glycans. Increased ionization temperatures and the use of methanol as a solvent further broadened the distribution, suggesting the enhanced conformational dynamics of the glycan ions. In contrast, sodium adduct ions yielded narrower distributions, implying that the interactions between sodium ions and glycans constrained structural flexibility. These results demonstrate that ionization parameters have a significant impact on glycan conformational behavior and mobility in the gas phase. This study provides insights into the analytical conditions for IM–MS measurements of glycans and highlights the utility of this method as a powerful tool for elucidating glycan structure and dynamics. 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

Bunch compactness (BC) is a complex, multi-trait characteristic that has been studied mostly in the context of wine grapes, with table grapes being scarcely considered. As these groups have marked phenotypic and genetic differences, including BC, the study of this trait is reported here using a genetically diverse collection of 116 Vitis vinifera L. cultivars and lines enriched for table grapes over two seasons. For this, 3D scanning-based morphological data were combined with ground measurements of 14 BC-related traits, observing high correlations among both approaches (R² > 0.90–0.97). The multivariate analysis suggests that the attributes ‘berries per bunch’, ‘berry weight and width’, and ‘bunch weight and length’ could be considered as the main descriptors for BC, optimizing evaluation times. Then, GWASs based on a set of 70,335 SNPs revealed that GBS analysis in this same population enabled the detection of several SNPs associated with different sub-traits, with a locus for ‘berries per bunch’ in chromosome (chr) 18 being the most prominent. Enrichment analysis of significant and frequent SNPs found simultaneously in several traits and seasons revealed the over-representation of discrete functions such as alpha-linolenic acid metabolism and glycan degradation. In summary, the utility of 3D automated phenotyping was validated for table grape backgrounds, and new SNPs and candidate genes associated with the BC trait were detected. The latter could eventually become a selection tool for grapevine breeding programs. Full article

This review summarizes recent findings on the diverse roles of bacterial sialidases in microbial biology. Bacterial sialidases, also known as neuraminidases, are exog α-lycosidases that cleave terminal sialic acid residues from a number of complex compounds designated as sialoglycoconjugates (glycoproteins, glycolipids and oligosaccharides). Metabolically, they are involved in sialic acid catabolism, providing energy, carbon and nitrogen sources. Catabolic degradation of sialic acids is a physiological feature that can be considered an important virulence factor in pathogenic microorganisms. Sialidases play a pivotal role in host–pathogen interactions and promotion of bacterial colonization. The activity of these enzymes enables bacterial adhesion, biofilm formation, tissue invasion, and also provides immune evasion by exposing cryptic receptors and modifying immune components. Many different perspectives are being developed for the potential application of sialidases. In the field of medicine, they are being explored as appropriate targets for antimicrobials, vaccines, diagnostic preparations and in tumor immunotherapy. In the field of enzymatic synthesis, they are used for the regioselective production of oligosaccharide analogs, enzymatic separation of isoenzymes and as a tool for structural analysis of sialylated glycans, among other applications. Full article

Background: Extra-intestinal pathogenic Escherichia coli (ExPEC) represents a major global public health challenge due to its ability to cause diverse clinical infections, including urinary tract infections, bacteremia, neonatal meningitis, and sepsis. The growing prevalence of multidrug-resistant (MDR) ExPEC strains, which rapidly erode antibiotic efficacy, underscores vaccine development as a critical priority. Bioconjugate vaccines have emerged as a promising approach to mitigate ExPEC-associated infections. Methods and Results: In this study, we utilized protein glycan coupling technology (PGCT) based on oligosaccharyltransferase (OST) PglL to engineer a tetravalent bioconjugate vaccine targeting four predominant ExPEC serotypes (O1, O2, O6, and O25). We conducted a series of experiments to demonstrate the efficacy of the conjugate vaccine in eliciting humoral immune responses and inducing the production of specific antibodies against Escherichia coli O1, O2, O6, or O25 serotypes. Conclusions: This work establishes the first application of the O-linked PGCT system for engineering bioconjugate vaccines against ExPEC infections. Full article

Phosphatidylinositol glycan class T (PIGT) is part of the glycosylphosphatidylinositol transamidase (GPI-TA) complex, crucial for various cell functions. Biallelic pathogenic variants in PIGT are associated with Multiple Congenital Anomalies-Hypotonia Seizures Syndrome 3 (MCAHS3), a rare neonatal hypotonia syndrome characterized by dysmorphic features and seizures. Diagnosing neonatal hypotonia, which has diverse congenital and acquired causes, is challenging, particularly in syndromic monogenic cases. Next-generation sequencing is essential for accurate diagnosis. This study reports a term newborn with hypotonia, dysmorphic features, seizures, and severe skeletal issues, including a humeral fracture at birth, consistent with MCAHS3. Trio whole exome sequencing (WES) analysis revealed a novel homozygous missense variant in PIGT, expanding the clinical spectrum of MCAHS3 and marking the first such case in the Thai population. The identified c.257A>G (p.His86Arg) variant manifests a severe MCAHS3 phenotype, as evidenced by reduced CD59 expression in western blot analysis, indicating impaired GPI-AP synthesis. Computational predictions suggest this mutation causes protein instability, potentially affecting GPI anchor attachment. While alkaline phosphatase (ALP), a GPI-AP crucial for skeletal mineralization, was elevated in this case, suggesting a late-stage GPI synthesis defect. The His86Arg mutation in PIGT may disrupt GPI-TA function, hindering proper protein attachment and leading to cleaved protein secretion. Further functional studies are needed to elucidate the impact of this mutation on PIGT function and MCAHS3 phenotypes. Full article

The glycosylation process plays a crucial role in the structural integrity and biological activity of glycoproteins, where glycans are attached to a protein backbone. There are many kinds of glycans, the most common being N-glycans, which can be arranged into three classes, that is, complex, hybrid, and high mannoses, forming a structurally very diverse set of polar compounds that are difficult to detect and separate. Most commonly, N-glycans are labeled before separation by charged or fluorescence tags for better MS or fluorescence detection, respectively. This study examines the influence of ionic strength and organic modifier selection on the separation of fluorescently labeled dextran ladders in Hydrophilic Interaction Liquid Chromatography (HILIC). Using a Glycan BEH Amide column and varying the ammonium formate buffer concentration along with acetonitrile and methanol ratios, we investigated analyte retention, separation efficiency, and post-column conductivity changes. Our findings reveal that changes in the ionic strength of the mobile phase do not contribute to changes in selectivity, neither when acetonitrile nor methanol were used as organic modifiers to the mobile phase. However, the addition of methanol significantly changes the separation mechanism where two different prevailing separations mechanisms can be identified. It was assumed that the addition of methanol influences the folding pattern of dextrans around the permanent positive charge on the added tag, which influences the changes of separation selectivity. This work presents a systematic approach to altering mobile phase composition (buffer concentration, organic modifier type) to control retention and selectivity in complex glycan analysis. The discovery that methanol significantly alters separation behavior provides a potential new method for refining HILIC separations of polar compounds. Full article