High-risk human papillomaviruses (HPVs) cause various cancers. While type-specific prophylactic vaccines are available, additional anti-viral strategies are highly desirable. Initial HPV cell entry involves receptor-switching induced by structural capsid modifications. These modifications are initiated by interactions with cellular heparan sulphates (HS), however, their molecular nature and functional consequences remain elusive. Combining virological assays with hydrogen/deuterium exchange mass spectrometry, and atomic force microscopy, we investigate the effect of capsid-HS binding and structural activation. We show how HS-induced structural activation requires a minimal HS-chain length and simultaneous engagement of several binding sites by a single HS molecule. This engagement introduces a pincer-like force that stabilizes the capsid in a conformation with extended capsomer linkers. It results in capsid enlargement and softening, thereby likely facilitating L1 proteolytic cleavage and subsequent L2-externalization, as needed for cell entry. Our data supports the further devising of prophylactic strategies against HPV infections.
- MeSH
- heparitinsulfát * metabolismus chemie MeSH
- infekce papilomavirem virologie MeSH
- internalizace viru * MeSH
- kapsida * metabolismus chemie MeSH
- lidé MeSH
- lidské papilomaviry MeSH
- lidský papilomavirus 16 metabolismus fyziologie MeSH
- mikroskopie atomárních sil * MeSH
- Papillomaviridae fyziologie MeSH
- polysacharidy metabolismus chemie MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- virové plášťové proteiny * metabolismus chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Mammalian spermatozoa have a surface covered with glycocalyx, consisting of heterogeneous glycoproteins and glycolipids. This complexity arises from diverse monosaccharides, distinct linkages, various isomeric glycans, branching levels, and saccharide sequences. The glycocalyx is synthesized by spermatozoa developing in the testis, and its subsequent alterations during their transit through the epididymis are a critical process for the sperm acquisition of fertilizing ability. In this study, we performed detailed analysis of the glycocalyx on the sperm surface of bull spermatozoa in relation to individual parts of the epididymis using a wide range (24) of lectins with specific carbohydrate binding preferences. Fluorescence analysis of intact sperm isolated from the bull epididymides was complemented by Western blot detection of protein extracts from the sperm plasma membrane fractions. Our experimental results revealed predominant sequential modification of bull sperm glycans with N-acetyllactosamine (LacNAc), followed by subsequent sialylation and fucosylation in a highly specific manner. Additionally, variations in the lectin detection on the sperm surface may indicate the acquisition or release of glycans or glycoproteins. Our study is the first to provide a complex analysis of the bull sperm glycocalyx modification during epididymal maturation.
- MeSH
- epididymis * metabolismus cytologie MeSH
- glykokalyx * metabolismus MeSH
- glykoproteiny metabolismus MeSH
- lektiny * metabolismus MeSH
- polysacharidy metabolismus MeSH
- skot MeSH
- spermie * metabolismus MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Prostate cancer (PCa) is the second most common cancer. In this paper, the isolation and properties of exosomes as potential novel liquid biopsy markers for early PCa liquid biopsy diagnosis are investigated using two prostate human cell lines, i.e., benign (control) cell line RWPE1 and carcinoma cell line 22Rv1. Exosomes produced by both cell lines are characterised by various methods including nanoparticle-tracking analysis, dynamic light scattering, scanning electron microscopy and atomic force microscopy. In addition, surface plasmon resonance (SPR) is used to study three different receptors on the exosomal surface (CD63, CD81 and prostate-specific membrane antigen-PMSA), implementing monoclonal antibodies and identifying the type of glycans present on the surface of exosomes using lectins (glycan-recognising proteins). Electrochemical analysis is used to understand the interfacial properties of exosomes. The results indicate that cancerous exosomes are smaller, are produced at higher concentrations, and exhibit more nega tive zeta potential than the control exosomes. The SPR experiments confirm that negatively charged α-2,3- and α-2,6-sialic acid-containing glycans are found in greater abundance on carcinoma exosomes, whereas bisecting and branched glycans are more abundant in the control exosomes. The SPR results also show that a sandwich antibody/exosomes/lectins configuration could be constructed for effective glycoprofiling of exosomes as a novel liquid biopsy marker.
- MeSH
- exozómy * chemie MeSH
- karcinom * metabolismus patologie MeSH
- lektiny analýza metabolismus MeSH
- lidé MeSH
- polysacharidy analýza metabolismus MeSH
- tekutá biopsie MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Breast cancer is a highly heterogeneous disease. Its intrinsic subtype classification for diagnosis and choice of therapy traditionally relies on the presence of characteristic receptors. Unfortunately, this classification is often not sufficient for precise prediction of disease prognosis and treatment efficacy. The N-glycan profiles of 145 tumors and 10 healthy breast tissues were determined using Matrix-Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry. The tumor samples were classified into Mucinous, Lobular, No-Special-Type, Human Epidermal Growth Factor 2 + , and Triple-Negative Breast Cancer subtypes. Statistical analysis was conducted using the reproducibility-optimized test statistic software package in R, and the Wilcoxon rank sum test with continuity correction. In total, 92 N-glycans were detected and quantified, with 59 consistently observed in over half of the samples. Significant variations in N-glycan signals were found among subtypes. Mucinous tumor samples exhibited the most distinct changes, with 28 significantly altered N-glycan signals. Increased levels of tri- and tetra-antennary N-glycans were notably present in this subtype. Triple-Negative Breast Cancer showed more N-glycans with additional mannose units, a factor associated with cancer progression. Individual N-glycans differentiated Human Epidermal Growth Factor 2 + , No-Special-Type, and Lobular cancers, whereas lower fucosylation and branching levels were found in N-glycans significantly increased in Luminal subtypes (Lobular and No-Special-Type tumors). Clinically normal breast tissues featured a higher abundance of signals corresponding to N-glycans with bisecting moiety. This research confirms that histologically distinct breast cancer subtypes have a quantitatively unique set of N-glycans linked to clinical parameters like tumor size, proliferative rate, lymphovascular invasion, and metastases to lymph nodes. The presented results provide novel information that N-glycan profiling could accurately classify human breast cancer samples, offer stratification of patients, and ongoing disease monitoring.
Galectins are proteins of the family of human lectins. By binding terminal galactose units of cell surface glycans, they moderate biological and pathological processes such as cell signaling, cell adhesion, apoptosis, fibrosis, carcinogenesis, and metabolic disorders. The binding of monovalent glycans to galectins is usually relatively weak. Therefore, the presentation of carbohydrate ligands on multivalent scaffolds can efficiently increase and/or discriminate the affinity of the glycoconjugate to different galectins. A library of glycoclusters and glycodendrimers with various structural presentations of the common functionalized N-acetyllactosamine ligand was prepared to evaluate how the mode of presentation affects the affinity and selectivity to the two most abundant galectins, galectin-1 (Gal-1) and galectin-3 (Gal-3). In addition, the effect of a one- to two-unit carbohydrate spacer on the affinity of the glycoconjugates was determined. A new design of the biolayer interferometry (BLI) method with specific AVI-tagged constructs was used to determine the affinity to galectins, and compared with the gold-standard method of isothermal titration calorimetry (ITC). This study reveals new routes to low nanomolar glycoconjugate inhibitors of galectins of interest for biomedical research.
- MeSH
- galektiny * metabolismus MeSH
- glykokonjugáty * farmakologie chemie MeSH
- lidé MeSH
- ligandy MeSH
- polysacharidy metabolismus MeSH
- sacharidy chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Protein glycosylation is one of the most common PTMs and many cell surface receptors, extracellular proteins, and biopharmaceuticals are glycosylated. However, HDX-MS analysis of such important glycoproteins has so far been limited by difficulties in determining the HDX of the protein segments that contain glycans. We have developed a column containing immobilized PNGase Rc (from Rudaea cellulosilytica) that can readily be implemented into a conventional HDX-MS setup to allow improved analysis of glycoproteins. We show that HDX-MS with the PNGase Rc column enables efficient online removal of N-linked glycans and the determination of the HDX of glycosylated regions in several complex glycoproteins. Additionally, we use the PNGase Rc column to perform a comprehensive HDX-MS mapping of the binding epitope of a mAb to c-Met, a complex glycoprotein drug target. Importantly, the column retains high activity in the presence of common quench-buffer additives like TCEP and urea and performed consistent across 114 days of extensive use. Overall, our work shows that HDX-MS with the integrated PNGase Rc column can enable fast and efficient online deglycosylation at harsh quench conditions to provide comprehensive analysis of complex glycoproteins.
- MeSH
- glykopeptidasa MeSH
- glykoproteiny * analýza MeSH
- glykosylace MeSH
- polysacharidy * metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
Polyglutamic acid (PGA), a protein in the mucilage of PGA-producing Bacillus spp., has expected applications in medical and biotechnological industries. Although the Bacillaceae family contains over 100 genera, research on bacterial PGA has exclusively focused on the genus Bacillus, especially B. subtilis var. natto and B. licheniformis. In the present study, indigenous Bacillaceae family strains were isolated from withered leaves and soil samples and screened for PGA production. As a result of the screening, the strain 8h was found to produce a mucilage possessing greater viscosity than PGA of B. subtilis var. natto (natto PGA). Biochemical analyses revealed that the 8h mucilage contains 63% protein and 37% polysaccharide, while mucilage of B. subtilis var. natto is composed of 61% protein and 39% polysaccharide. The most plentiful amino acid in 8h mucilage protein was glutamate (43%, mol/mol), which is similar to that of natto PGA, suggesting that it possesses characteristics of PGA. Although natto mucilage contains fructan, glucan was found as the polysaccharide of 8h mucilage. While phylogenetic studies indicated that the strain 8h belongs to Peribacillus simplex, the yield of the viscous mucilage by strain 8h was significantly higher than P. simplex type strain, suggesting that 8h is a mucilage-overproducing strain of P. simplex. Interestingly, 8h mucilage protein was found to contain more hydrophobic amino acid residues than natto PGA, suggesting that its amphiphilicity is suitable as a drug carrier and adjuvant. The present study is the first report of viscous mucilage and PGA-like protein produced by the genus Peribacillus.
INTRODUCTION: N-glycosylation is a ubiquitous and variable posttranslational modification that regulates physiological functions of secretory and membrane-associated proteins and the dysregulation of glycosylation pathways is often associated with cancer growth and metastasis. Prostate-specific membrane antigen (PSMA) is an established biomarker for prostate cancer imaging and therapy. METHODS: Mass spectrometry was used to analyze the distribution of the site-specific glycoforms of PSMA in insect, human embryonic kidney, and prostate cancer cells, and in prostate tissue upon immunoaffinity enrichment. RESULTS: While recombinant PSMA expressed in insect cells was decorated mainly by paucimannose and high mannose glycans, complex, hybrid, and high mannose glycans were detected in samples from human cells and tissue. We noted an interesting spatial distribution of the glycoforms on the PSMA surface-high mannose glycans were the dominant glycoforms at the N459, N476, and N638 sequons facing the plasma membrane, while the N121, N195, and N336 sites, located at the exposed apical PSMA domain, carried primarily complex glycans. The presence of high mannose glycoforms at the former sequons likely results from the limited access of enzymes of the glycosynthetic pathway required for the synthesis of the complex structures. In line with the limited accessibility of membrane-proximal sites, no glycosylation was observed at the N51 site positioned closest to the membrane. CONCLUSIONS: Our study presents initial descriptive analysis of the glycoforms of PSMA observed in cell lines and in prostate tissue. It will hopefully stimulate further research into PSMA glycoforms in the context of tumor staging, noninvasive detection of prostate tumors, and the impact of glycoforms on physicochemical and enzymatic characteristics of PSMA in a tissue-specific manner.
- MeSH
- antigeny povrchové metabolismus MeSH
- buněčné linie MeSH
- glutamátkarboxypeptidasa II metabolismus MeSH
- glykosylace MeSH
- hmotnostní spektrometrie metody MeSH
- lidé MeSH
- nádorové biomarkery analýza MeSH
- nádory prostaty * metabolismus patologie MeSH
- polysacharidy * klasifikace metabolismus MeSH
- posttranslační úpravy proteinů MeSH
- prostata * enzymologie metabolismus patologie MeSH
- staging nádorů MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Carbohydrates form one of the major groups of biological macromolecules in living organisms. Many biological processes including protein folding, stability, immune response, and receptor activation are regulated by glycosylation. Fucosylation of proteins regulates such processes and is associated with various diseases including autoimmunity and cancer. Mass spectrometry efficiently identifies structures of fucosylated glycans or sites of core fucosylated N-glycopeptides but quantification of the glycopeptides remains less explored. We performed experiments that facilitate quantitative analysis of the core fucosylation of proteins with partial structural resolution of the glycans and we present results of the mass spectrometric SWATH-type DIA analysis of relative abundances of the core fucosylated glycoforms of 45 glycopeptides to their nonfucosylated glycoforms derived from 18 serum proteins in liver disease of different etiologies. Our results show that a combination of soft fragmentation with exoglycosidases is efficient at the assignment and quantification of the core fucosylated N-glycoforms at specific sites of protein attachment. In addition, our results show that disease-associated changes in core fucosylation are peptide-dependent and further differ by branching of the core fucosylated glycans. Further studies are needed to verify whether tri- and tetra-antennary core fucosylated glycopeptides could be used as markers of liver disease progression.
- MeSH
- biologické markery metabolismus MeSH
- chromatografie kapalinová metody MeSH
- fukosa metabolismus MeSH
- glykopeptidy metabolismus MeSH
- glykosidhydrolasy * MeSH
- glykosylace MeSH
- jaterní cirhóza diagnóza metabolismus MeSH
- lidé MeSH
- polysacharidy metabolismus MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Mucosal surfaces are colonized by highly diverse commensal microbiota. Coating with secretory IgA (SIgA) promotes the survival of commensal bacteria while it inhibits the invasion by pathogens. Bacterial coating could be mediated by antigen-specific SIgA recognition, polyreactivity, and/or by the SIgA-associated glycans. In contrast to many in vitro studies, only a few reported the effect of SIgA glycans in vivo. Here, we used a germ-free antibody-free newborn piglets model to compare the protective effect of SIgA, SIgA with enzymatically removed N-glycans, Fab, and Fc containing the secretory component (Fc-SC) during oral necrotoxigenic E. coli O55 challenge. SIgA, Fab, and Fc-SC were protective, whereas removal of N-glycans from SIgA reduced SIgA-mediated protection as demonstrated by piglets' intestinal histology, clinical status, and survival. In vitro analyses indicated that deglycosylation of SIgA did not reduce agglutination of E. coli O55. These findings highlight the role of SIgA-associated N-glycans in protection. Further structural studies of SIgA-associated glycans would lead to the identification of those involved in the species-specific inhibition of attachment to corresponding epithelial cells.
- MeSH
- aglutinace MeSH
- Escherichia coli fyziologie MeSH
- glykosylace MeSH
- gnotobiologické modely MeSH
- imunoglobulin A sekreční metabolismus MeSH
- imunoglobuliny - Fab fragmenty metabolismus MeSH
- infekce vyvolané Escherichia coli imunologie MeSH
- jednořetězcové protilátky metabolismus MeSH
- novorozená zvířata MeSH
- odolnost vůči nemocem MeSH
- polysacharidy metabolismus MeSH
- prasata MeSH
- těhotenství MeSH
- zvířata MeSH
- Check Tag
- těhotenství MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
