Search Results (611)

The Candida parapsilosis complex (CPC), comprising Candida parapsilosis sensu stricto, Candida orthopsilosis, and Candida metapsilosis, is a significant cause of nosocomial candidemia worldwide. Because species-specific drug resistance profiles vary, accurate identification is clinically crucial; however, mainstream molecular diagnostics require expensive instrumentation. To establish a baseline for simple, cost-effective immunochemical diagnostics, we elucidated the cell wall N-linked mannan structures across representative CPC strains. Intact N-linked mannan cores were isolated via the classical Fehling method, which efficiently eliminated confounding O-linked glycans. Contrary to the historical paradigm suggesting a lack of branched side chains in C. parapsilosis, two-dimensional ¹H-¹H total correlation spectroscopy (2D-TOCSY) clearly identified a unique di-substituted branching residue, Manα1-2Manα1-3(Manα1-6)Man, corresponding to antigenic factor 4. Acetolysis and gel filtration demonstrated that the side chains ranged from a degree of polymerization (DP) of 2 to 6, with a tetrasaccharide serving as the structural mainstay. These results redefine the antigenic factor profile of C. parapsilosis as (1, 4, 13b), distinguishing it from C. orthopsilosis and C. metapsilosis (1, 13b). By integrating these N-linked profiles with our recent O-linked data, this study delineates the comprehensive cell wall surface architectures among the CPC. This framework provides a critical molecular foundation for the future development of rapid serological identification kits, while enhancing our understanding of evolutionary diversity and biosynthetic pathways within pathogenic yeasts. Full article

ENZ-124, the first approved cetuximab biosimilar by Indian health authorities, was developed using the Chinese hamster ovary cell line, whereas the SP2/0 cell line was used for developing cetuximab originator (Erbitux^®). Hence, this assessment aimed to evaluate the physiochemical, structural, and biological characterization of four batches each of Cetuxa^®, Lupitux^® and cetuximab originator. Analytical methods like peptide mapping (liquid chromatography–tandem mass spectrometry [LC-MS/MS]), glycan mapping (liquid chromatography–fluorescence detection), isoform analysis (hydrophobic interaction chromatography [HIC]-high-performance liquid chromatography), charge variant analysis (capillary electrophoresis coupled with mass spectrometry), FcγRIIIa binding assays, cell-based antibody-dependent cell-mediated cytotoxicity (ADCC) measurement, and host cell protein analysis (LC-MS/MS) were utilized for analysis. Variations in physicochemical and biological attributes were observed between cetuximab originator and its biosimilars. HIC analysis revealed C-terminal heterogeneity and increased hydrophobicity in biosimilars, that with the different N-glycan profile affected charge distribution. Charge variant analysis indicated a more acidic profile for the biosimilars. The biosimilars showed higher oxidation levels and misincorporation. The biosimilars also showed an enhanced FcγRIIIa binding affinity and in vitro ADCC activation compared with cetuximab originator. The study outcomes highlight the ability to differentiate cetuximab originator from its biosimilars Cetuxa^® and Lupitux^® based on the observed physicochemical and biological differences. Full article

Small biomolecules provide important information on cellular metabolism, signaling and disease-associated molecular changes. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) enables rapid, high-throughput and spatially resolved molecular analysis, but its application to small biomolecules is often limited by poor ionization, low-mass background interference, ion suppression and insufficient structural information. To address these analytical challenges, this review summarizes chemical derivatization strategies that improve MALDI-MS analysis of small biomolecules. The representative strategies are discussed according to reaction mode and target functional group, including solution-phase, on-target, on-tissue, reactive-matrix-assisted and photo-/in-source approaches, with emphasis on amine-, carbonyl-, carboxyl- and double-bond-containing biomolecules. Chemical derivatization improves MALDI-MS performance by selectively modifying target functional groups, introducing charged or ionizable tags, enhancing molecular discrimination and providing additional structural information. These strategies have expanded the detection and annotation of neuroactive amines, carbonyl compounds, glycans, carboxylic acid metabolites and lipid double-bond isomers, while also extending MALDI-MS analysis to other specific functional groups, natural products and drug-related molecules. Overall, chemical derivatization is an important approach for improving the sensitivity, selectivity, structural annotation and analytical coverage of MALDI-MS-based small biomolecule analysis. Future developments should further address reaction selectivity, spatial fidelity, quantitative reliability and confident identification of derivatization products. Full article

Keyhole limpet hemocyanin (KLH) is a large copper-containing glycoprotein derived from the marine gastropod Megathura crenulata. Originally functioning as an oxygen transport molecule, KLH has gained considerable attention in biomedical research due to its exceptional immunogenic and immunostimulatory properties. Its complex quaternary structure, extensive glycosylation, and xenogeneic origin contribute to its ability to induce robust humoral and cellular immune responses in mammals without significant toxicity. These characteristics have established KLH as one of the most widely used carrier proteins in vaccine development and as a valuable model antigen for the investigation of adaptive immune responses. This review summarizes current knowledge on the biological origin, molecular structure, biosynthesis, and post-translational processing of KLH, with particular emphasis on its unique glycan architecture and its contribution to immunogenicity. Advances in glycomic and structural analyses have revealed an extraordinary diversity of N-linked glycans that distinguish KLH from mammalian glycoproteins and play a central role in immune recognition. The review further discusses methods for KLH isolation, purification, and characterization, as well as its application in experimental and clinical immunology as a standardized tool for assessing antigen-specific immune responses. In addition, the therapeutic and translational potential of KLH is examined across multiple biomedical fields. Particular attention is given to its use as a carrier protein in conjugate vaccines, its role in cancer immunotherapy, and its emerging applications in the development of vaccines and immunotherapeutic strategies targeting neurodegenerative diseases, atherosclerosis, and substance use disorders. Collectively, the available evidence highlights KLH as a unique marine-derived biomolecule that bridges glycobiology, immunology, and translational medicine, and continues to serve as an important platform for the development of next-generation immunotherapeutics and vaccine technologies. Full article

Glycosylation is a critical determinant of the efficacy, stability, and pharmacological behavior of therapeutic proteins. R27T, an engineered variant of interferon-β1a, contains two N-glycosylation sites (Asn25 and Asn80), increasing its structural complexity and analytical requirements. In this study, we performed comprehensive total and site-specific glycan profiling of R27T using complementary analytical approaches. For total glycan analysis, the released N-glycans were fluorescently labeled with procainamide, providing enhanced sensitivity and broader glycan coverage compared with conventional 2-aminobenzamide labeling. Site-specific glycan profiling was performed by liquid chromatography–tandem mass spectrometry (LC–MS/MS)-based peptide mapping. Protease digestion conditions were optimized to improve recovery of site-specific glycopeptides, with chymotrypsin identified as the most effective enzyme for resolving glycopeptides from individual glycosylation sites. Total glycan distributions reconstructed from peptide-mapping data were compared with fluorescence-based glycan profiling, showing that total and site-specific glycan data can be effectively combined. Minor discrepancies were observed depending on glycan structure, mainly due to differences in ionization efficiency. Distinct glycan distributions were observed between the two N-glycosylation sites of R27T. Molecular modeling further suggested that the additional glycan at Asn25 may enhance structural stability and receptor-binding affinity. These results demonstrate an integrative strategy for accurate glycan characterization in multi-site glycoproteins relevant to biotherapeutic development. Full article

Biofilms contribute significantly to the persistence and transmission of Campylobacter jejuni, yet enzymatic strategies for disrupting these structures remain underexplored. In this study, we evaluated the activity of a glycosyl hydrolase, CAase, against both planktonic and biofilm-associated C. jejuni. CAase showed no inhibitory effect on planktonic growth at concentrations up to 1 mg/mL. However, at 0.1 mg/mL, CAase reduced mature biofilm biomass by more than 93% in the wild-type strain and produced similar reductions in a luxS mutant, demonstrating substantial degradation of conserved glycan components within the extracellular polymeric substance (EPS). Scanning electron microscopy (SEM) imaging revealed substantial extracellular matrix loss, structural disruption, and altered cell morphology following CAase treatment. Rheological measurements demonstrated marked decreases in kinematic viscosity, further reflecting weakened biofilm cohesion. Together, these results indicate that CAase effectively disrupts multiple components of C. jejuni biofilm architecture despite lacking direct antimicrobial activity. This level of biofilm removal suggests potential utility in poultry-processing or surface-sanitation applications where targeted enzymatic disruption could enhance cleaning efficacy. Full article

N-linked glycans (N-glycans) are complex carbohydrate structures covalently attached to the asparagine residues of glycoproteins and play a substantial role in protein folding, stability and biological activity. One of the major challenges in glycomics is the enzymatic release of intact N-glycans from native glycoproteins, as conventional deglycosylation approaches often require prior protein denaturation and exhibit limited activity toward structurally diverse substrates. In this study, we recombinantly produced EndoBI-1, an endo-β-N-acetylglucosaminidase derived from Bifidobacterium longum subsp. infantis ATCC 15697, and investigated its N-glycan release activity on two structurally distinct glycoprotein substrates: bovine lactoferrin (bLF), an animal-derived glycoprotein, and soy protein, a plant-derived glycoprotein. EndoBI-1 was produced using an in vivo cloning approach and purified to homogeneity, as validated by SDS-PAGE analysis. The released N-glycan profiles of each substrate were characterized by HILIC-FLD-QTOF-MS/MS analysis, which showed different glycan compositions depending on the origin: complex-type and sialylated N-glycans were the major structures in the bLF derived fractions, while the fractions derived from soy protein mainly contained high-mannose-type structures. Together, these results demonstrate the capacity of EndoBI-1 to release structurally intact N-glycans from both animal- and plant-derived glycoproteins under native, non-denaturing conditions, establishing this enzyme as a biocatalytic tool for the preparative-scale production of bioactive N-glycans from diverse protein sources. Full article

As a vital post-translational modification of potato tuber proteins, N-glycosylation directly regulates protein structural stability, biological activity and processing properties. N-glycome profiles of tuber storage proteins exhibit cultivar-specific characteristics, but the differential glycosylation across potato germplasms has not been fully elucidated. In this work, the major tuber patatin glycoprotein was purified from three potato cultivars as research material to investigate their N-glycosylation features and inter-cultivar variations. N-glycans were released via nonreductive chemical cleavage and derivatized with PMP, followed by qualitative and quantitative profiling using liquid chromatography-tandem mass spectrometry (LC-UV-MS/MS). In total, 11, 6 and 3 distinct N-glycan structures were identified from the three tested cultivars. Two novel glycan structures, Man₃XylGlcNAc₂ and Man₄XylGlcNAc₂, were identified in this study. The relative abundances of Man₃XylFucGlcNAc₂ and Man₃XylGlcNAc₂ exhibited highly significant inter-cultivar differences (p < 0.01). Notably, patatin carried unique N-glycans modified with exclusive α1,3-fucosylation, and overall glycan composition and relative abundance varied markedly across the three cultivars. This study systematically elucidates the effects of potato cultivar germplasm on patatin N-glycosylation and analyzes the unique glycosylation signatures of patatin from the three varieties. The obtained glycomic dataset provides fundamental theoretical support for germplasm screening, functional modification of potato storage proteins, and the optimization of potato deep-processing. Full article

Glycans attached to host glycoproteins play an important role in regulating oral microbial colonization and maintaining community balance. Our previous studies revealed that children exhibit lower levels of α2-3 sialylated glycan structures (SAα2-3Gal) than adults, raising the possibility that this age-associated glycan deficiency contributes to the heightened cariogenicity of Streptococcus mutans. In this study, sialylated glycoproteins (Sia-GP) and corresponding desialylated controls (DeSia-GP and α2,3-DeSia-GP) were prepared from bovine milk-derived glycoproteins to investigate the functional contribution of SAα2-3Gal. Their effects on S. mutans were evaluated by assessing bacterial growth, acid production, biofilm formation and extracellular polysaccharide synthesis, while a human oral keratinocytes (HOK cells) infection model was used to examine epithelial cell viability, wound healing, and infection-associated inflammatory responses. The results showed that Sia-GP exerted only a transient inhibitory effect on early bacterial growth without affecting final biomass, but significantly attenuated acidogenic activity, biofilm formation, and extracellular polysaccharides production in a concentration-dependent manner. These inhibitory effects were markedly attenuated following removal of α2-3-linked sialic acids, demonstrating the essential role of SAα2-3Gal. In addition, Sia-GP alleviated S. mutans-induced cellular damage in HOK cells by improving cell viability, colony formation, and migration, while suppressing infection-associated inflammatory signaling. Collectively, these findings demonstrate that SAα2-3Gal effectively limits multiple virulence traits of S. mutans and attenuates S. mutans-induced damage in oral epithelial cells. This study provides mechanistic insights into glycan-mediated regulation of host–microbe interactions and suggests that the naturally low abundance of SAα2-3Gal in children may increase their susceptibility to S. mutans infection and caries development. Full article

Soil mulching influences soil organic carbon (SOC) dynamics and microbial communities, yet the functional potential linking these factors remains unclear. This two-year field study compared biodegradable liquid film (BLF, C:N = 26:1, 161 kg C·ha⁻¹) and grapevine branch mulch (GBM, C:N = 51:1, 2790 kg C·ha⁻¹) applied in-row in a vineyard, with clean tillage as control. The Vitis vinifera cv. Meili was used as the test material, SOC fractions were determined and metagenomic sequencing was performed. The results showed that GBM had the highest SOC content and significantly increased the levels of total organic carbon, all five labile fractions, and the three recalcitrant fractions. BLF significantly increased the levels of recalcitrant fractions, while its effect on labile fractions varied by year. Metagenomic analysis revealed that the two mulching treatments significantly influenced the abundances of Acidobacteria, Verrucomicrobia, and Bacteroidetes. Redundancy analysis identified soil moisture, pH, SOC, and total nitrogen as key drivers of community structure. Mulching downregulated carbon fixation and methane metabolism genes but upregulated carbohydrate metabolism pathways, including O-glycan biosynthesis, which correlated positively with SOC. Glycosyl transferases were the dominant carbohydrate-active enzymes across all treatments. These results demonstrate that GBM and BLF differentially affect SOC fractions and microbial functional traits, providing empirical evidence for mulch selection in vineyard carbon management. Full article

Background/Objectives: Glycosylation can affect vaccine antigen structure and function, making site-specific glycan characterization relevant to antigen quality and comparability. However, quantitative approaches for comparing glycan microheterogeneity remain limited. This study evaluated the utility of the glycopeptide abundance distribution spectra framework for measuring similarity among site-specific glycosylation profiles in vaccines and antigen reference reagents across manufacturing conditions. Methods: Intact N-linked glycopeptides were characterized by nanoflow liquid chromatography–tandem mass spectrometry with stepped-energy fragmentation. Products included monovalent and quadrivalent influenza antigens produced in embryonated eggs, Madin–Darby canine kidney cells, or Spodoptera frugiperda cells, together with a SARS-CoV-2 spike vaccine produced in Spodoptera frugiperda cells and a Chinese hamster ovary cell-produced varicella-zoster virus glycoprotein E vaccine. Site-specific glycan distributions were represented as distribution spectra and compared using NIST MS Search software. Dot-product scores ranging from 0 to 999 quantified similarity. Results: Across measured glycosylation sites, distributions clustered into six recurrent classes. Similarity was high for replicate analyses, conserved influenza components across annual formulations, and matched components from different suppliers within the same production platform (similarity scores = 978, 961, and 960, respectively). Similarity was lower between sites within the same protein, between influenza strains, and between production sources (similarity scores = 554, 540, and 209, respectively). Among production-source comparisons, egg- and Madin–Darby canine kidney-derived profiles were most similar, and the overall ordering of glycosylation similarity was consistent with broad phylogenetic relatedness among production hosts. Conclusions: Distribution spectra-based similarity scoring of vaccine glycoproteins provides a quantitative, reusable approach for documenting site-specific glycosylation microheterogeneity. Using this method, we can conclude that production source is the dominant contributor to variation, whereas replicates, annual formulations, and suppliers within the same production platform are highly consistent. Full article

Glycosyltransferases often contain multiple structural modules that contribute to substrate recognition, catalytic coordination, and higher-order molecular organization. However, how multidomain glycosyltransferases dynamically organize their catalytic domains in solution remains poorly understood. Here, we investigated the assembly states and conformational dynamics of POMGNT2, LARGE1, K4CP, and L137 using high-speed atomic force microscopy (HS-AFM) integrated with complementary solution biophysical analyses. This multi-technique approach extends previous static structural studies by enabling a parallel comparison of solution-state dynamics among multiple glycosyltransferases. POMGNT2 formed a stable dimeric architecture with limited large-scale conformational fluctuation, consistent with its role in site-selective substrate recognition. In contrast, LARGE1 and K4CP exhibited concentration-dependent and heterogeneous assembly behavior. K4CP displayed pronounced open–closed interdomain motion and a tendency toward more compact conformations in the presence of substrate, suggesting dynamic catalytic-domain reorganization during glycan elongation. By comparison, the mimivirus glycosyltransferase candidate L137 predominantly behaved as a monomeric species under the tested conditions. These findings demonstrate that multidomain glycosyltransferases employ diverse dynamic organizational strategies ranging from rigid recognition architectures to highly flexible and reversible catalytic assemblies. Our results further suggest that glycosyltransferase function is governed not only by catalytic-domain structure, but also by dynamic conformational coordination adapted to distinct catalytic demands. Full article

Congenital disorders of glycosylation (CDG) are a group of inherited metabolic diseases rapidly growing due to the discovery of new subtypes. As with many genetic conditions, their diagnosis can be challenging, impairing proper patient care and causing additional suffering to patients and their families. We have developed an N-glycomics strategy that can provide insightful information towards diagnosing CDG type II (CDG-II). N-glycans released from the plasma of healthy individuals were labeled with deuterated iodomethane, mixed with samples from known or suspected CDG-II individuals, which were derivatized with standard iodomethane, and analyzed by liquid chromatography–mass spectrometry. After identification, relative quantification of 65 glycans was performed, revealing considerable alterations in the N-glycome of several patients. Notably, reduced fucosylation was observed in patients with FUT8-CDG and SLC35C1-CDG. Additionally, individuals with mutations in the MAN1B1 gene exhibited increased amounts of hybrid and oligomannosidic structures, whereas patients with the Golgi homeostasis disorders COG1-CDG and ATP6V0A2-CDG presented marked increases in hypogalactosylated and hyposialylated structures. Multivariate statistical analysis indicated two undiagnosed patients with alterations similar to ATP6V0A2-CDG patients and another two with a profile similar to MAN1B1-CDG patients. Genetic sequencing (targeted gene panel or whole exome sequencing) of these undiagnosed patients revealed variants in the corresponding genes, confirming the diagnosis obtained from the N-glycomics analysis. Our results demonstrate how the analysis of total plasma N-glycans can be used to identify metabolic disorders and diagnose conditions based on their molecular effects on the glycoproteome. Full article

Human anti-αGal antibodies (Abs), known for their marked polyreactivity, have been detected bound to the gray matter of the brains of Alzheimer’s disease (AD) patients, although their targets were unclear. Since αGal is a strictly xenogenic antigen absent in humans, this observation raised questions regarding the nature of the structures recognized by these antibodies. In this study, we investigated their potential interaction with gangliosides—glycan structures that are highly abundant in the central nervous system. Using a competitive inhibition ELISA, serum profiles of anti-αGal Abs isotypes and their indirect cross-reactivity with selected soluble gangliosides were analyzed in AD patients and healthy subjects (HSs). AD patients showed reduced levels of anti-αGal IgG and IgM, but increased IgA compared to HSs. Notably, pre-incubation with GM1, GM2, or GD1b did not reduce αGal–HSA binding in HS sera. In contrast, in AD sera, pre-incubation with GD1b reduced residual αGal–HSA binding for all antibody isotypes; additionally, GM1 inhibited IgM binding, and GM2 inhibited IgA binding. These results should therefore be interpreted as competitive inhibition patterns consistent with ganglioside-associated cross-reactivity rather than as direct evidence of antibody binding to immobilized gangliosides. Overall, the findings provide preliminary evidence that, in AD sera, a fraction of αGal–HSA-reactive antibodies can be competitively inhibited by selected gangliosides. This observation supports the presence of an altered humoral anti-carbohydrate signature in AD and identifies neuronal gangliosides as plausible candidate autologous targets that may help explain the previously reported binding of anti-αGal Abs to gray matter. However, given the indirect nature of the assay, these data should be considered hypothesis-generating and require confirmation by direct binding approaches. Full article

Malignant pleural mesothelioma (MPM) is an aggressive pleural tumor associated with asbestos exposure. Poor clinical outcome of MPM is often driven by late-stage diagnosis due to non-specific clinical presentation, similarity to pleural lesions (e.g., inflammatory changes, metastatic adenocarcinoma), and limitations of current diagnostic methods. We employed Fourier transform infrared (FTIR) spectroscopy combined with convolutional neural networks (CNNs) to analyze formalin-fixed paraffin-embedded (FFPE) pleural tissue samples from patients with MPM, metastatic adenocarcinoma, pleural inflammation, and normal (healthy) pleura. Glycan analysis of FFPE normal pleura and MPM was performed using ultra-high-performance liquid chromatography (UPLC) and mass spectrometry (MS). Our FTIR-spectral analysis uncovered a strong spectral fingerprint of MPM that was especially apparent in the region typical for C-O and C-C stretches as well as local symmetry region typical for deformation vibrations of CH₂ and C-OH groups, all appearing in carbohydrates. Our orthogonal validation of these findings through a targeted glycomics approach using UPLC confirmed that the MPM N-glycome exhibits a distinct fingerprint that distinguishes it from normal pleural tissue. Through utilization of MS for identifying the exact structures of differentially expressed N-glycan peaks, we also identified two high-mannose N-glycan structures that show a specific biomarker potential for MPM and need to be examined in future studies. Full article