Saccharides form one of the major constituents of biological macromolecules in living organisms. Many biological processes including protein folding, stability, immune response and receptor activation are regulated by glycosylation. In this work, we optimized a capillary electrophoresis method with capacitively coupled contactless conductivity detection for the separation of eight monosaccharides commonly found in glycoproteins, namely D-glucose, D-galactose, D-mannose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, D-fucose, N-acetylneuraminic acid, and D-xylose. A highly alkaline solution of 50 mM sodium hydroxide, 22.5 mM disodium phosphate, and 0.2 mM CTAB (pH 12.4) was used as a background electrolyte in a 10 μm id capillary. To achieve baseline separation of all analytes, a counter-directional pressure of -270 kPa was applied during the separation. The limits of detection of our method were below 7 μg/ml (i.e., 1.5 pg or 1 mg/g protein) and the limits of quantification were below 22 μg/ml (i.e., 5 pg or 3 mg/g protein). As a proof of concept of our methodology, we performed an analysis of monosaccharides released from fetuin glycoprotein by acid hydrolysis. The results show that, when combined with an appropriate pre-concentration technique, the developed method can be used as a monosaccharide profiling tool in glycoproteomics and complement the routinely used LC-MS/MS analysis.
- MeSH
- acetylgalaktosamin MeSH
- acetylglukosamin MeSH
- cetrimonium MeSH
- chromatografie kapalinová MeSH
- elektroforéza kapilární metody MeSH
- elektrolyty chemie MeSH
- fetuiny MeSH
- fosfáty MeSH
- fukosa MeSH
- galaktosa MeSH
- glukosa MeSH
- glykoproteiny chemie MeSH
- hydroxid sodný MeSH
- kyselina N-acetylneuraminová * MeSH
- mannosa MeSH
- monosacharidy * analýza MeSH
- tandemová hmotnostní spektrometrie MeSH
- xylosa MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Galectin-3 plays a crucial role in cancerogenesis; its targeting is a prospective pathway in cancer diagnostics and therapy. Multivalent presentation of glycans was shown to strongly increase the affinity of glycoconjugates to galectin-3. Further strengthening of interaction with galectin-3 may be accomplished using artificial glycomimetics with apt aryl substitutions. We established a new, as yet undescribed chemoenzymatic method to produce selective C-3-substituted N,N'-diacetyllactosamine glycomimetics and coupled them to human serum albumin. From a library of enzymes, only β-N-acetylhexosaminidase from Talaromyces flavus was able to efficiently synthesize the C-3-propargylated disaccharide. Various aryl residues were attached to the functionalized N,N'-diacetyllactosamine via click chemistry to assess the impact of the aromatic substitution. In ELISA-type assays with galectin-3, free glycomimetics exhibited up to 43-fold stronger inhibitory potency to Gal-3 than the lactose standard. Coupling to human serum albumin afforded multivalent neo-glycoproteins with up to 4209-fold increased inhibitory potency per glycan compared to the monovalent lactose standard. Surface plasmon resonance brought further information on the kinetics of galectin-3 inhibition. The potential of prepared neo-glycoproteins to target galectin-3 was demonstrated on colorectal adenocarcinoma DLD-1 cells. We investigated the uptake of neo-glycoproteins into cells and observed limited non-specific transport into the cytoplasm. Therefore, neo-glycoproteins primarily act as efficient scavengers of exogenous galectin-3 of cancer cells, inhibiting its interaction with the cell surface, and protecting T-lymphocytes against galectin-3-induced apoptosis. The present neo-glycoproteins combine the advantage of a straightforward synthesis, selectivity, non-toxicity, and high efficiency for targeting exogenous galectin-3, with possible application in the immunomodulatory treatment of galectin-3-overexpressing cancers.
- MeSH
- biomimetické materiály chemická syntéza chemie farmakologie MeSH
- galektiny antagonisté a inhibitory genetika metabolismus MeSH
- glykoproteiny chemie metabolismus MeSH
- kinetika MeSH
- krevní proteiny antagonisté a inhibitory genetika metabolismus MeSH
- lidé MeSH
- molekulární struktura MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Tick-borne encephalitis virus (TBEV) is the causative agent of severe human neuroinfections that most commonly occur after a tick bite. N-Glycosylation of the TBEV envelope (E) glycoprotein is critical for virus egress in mammalian cells, but not in tick cells. In addition, glycans have been reported to mask specific antigenic sites from recognition by neutralizing antibodies. In this regard, the main purpose of our study was to investigate the profile of N-glycans linked to the E protein of TBEV when grown in human neuronal cells and compare it to the profile of virus grown in tick cells. Mass spectrometric analysis revealed significant differences in these profiles. High-mannose glycan with five mannose residues (Man5GlcNAc2), a complex biantennary galactosylated structure with core fucose (Gal2GlcNAc2Man3GlcNAc2Fuc), and a group of hybrid glycans with the composition Gal0-1GlcNAc1Man3-5GlcNAc2Fuc0-1 were confirmed as the main asparagine-linked oligosaccharides on the surface of TBEV derived from human neuronal cells. The observed pattern was supported by examination of the glycopeptides, providing additional information about the glycosylation site in the E protein. In contrast, the profile of TBEV grown in tick cells showed that paucimannose (Man3-4 GlcNAc2Fuc0-1) and high-mannose structures with five and six mannoses (Man5-6GlcNAc2) were major glycans on the viral surface. The reported results complement existing crystallography and cryoelectron tomography data on the E protein structure and could be instrumental for designing carbohydrate-binding antiviral agents active against TBEV.
- MeSH
- glykoproteiny chemie metabolismus MeSH
- glykosylace MeSH
- klíšťata virologie MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- proteiny virového obalu chemie metabolismus MeSH
- sekvence aminokyselin MeSH
- viry klíšťové encefalitidy růst a vývoj metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Lepidopteran silk is a complex assembly of proteins produced by a pair of highly specialized labial glands called silk glands. Silk composition has been examined only in a handful of species. Here we report on the analysis of silk gland-specific transcriptomes from three developmental stages of the greater wax moth, Galleria mellonella, combined with proteomics, Edman microsequencing and northern blot analysis. In addition to the genes known earlier, we identified twenty seven candidate cDNAs predicted to encode secretory proteins, which may represent novel silk components. Eight were verified by proteomic analysis or microsequencing, and several others were confirmed by similarity with known silk genes and their expression patterns. Our results revealed that most candidates encode abundant secreted proteins produced by middle silk glands including ten sericins, two seroins, one or more mucins, and several sequences without apparent similarity to known proteins. We did not detect any novel PSG-specific protein, confirming that there are only three fibroin subunits. Our data not only show that the number of sericin genes in the greater wax moth is higher than in other species thus far examined, but also the total content of soluble proteins in silk is twice as high in G. mellonella than in B. mori or A. yamamai. Our data will serve as a foundation for future identification and evolutionary analysis of silk proteins in the Lepidoptera.
- MeSH
- fylogeneze MeSH
- glykoproteiny chemie genetika metabolismus MeSH
- hedvábí genetika metabolismus MeSH
- hmyzí proteiny chemie genetika metabolismus MeSH
- larva genetika růst a vývoj metabolismus MeSH
- muciny chemie genetika metabolismus MeSH
- můry genetika růst a vývoj metabolismus MeSH
- proteom * MeSH
- sekvence aminokyselin MeSH
- sekvence nukleotidů MeSH
- sekvenční seřazení MeSH
- sericiny chemie genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Rubroboletus satanas (Lenz) Kuan Zhao & Zhu L. Yang, known as Boletus satanas (Lenz) until 2014, and commonly known as the Devil´s bolete or Satan´s bolete, is a basidiomycete mushroom of the bolete family. Grows in mixed woodlands in the southern, warmer regions of Europe and North America. Satan´s bolete generally regarded as a poisonous mushroom, with predominantly gastrointestinal symptoms of nausea and violent vomiting occurring if eaten raw or isufficiently heat-treated. The toxicity of the mushroom corresponds to a toxic protein called bolesatine. Bolesatine is a toxic glycoprotein which has been shown to inhibit protein synthesis in cell-free systems and cell culture and is toxic to rodents. Biology, chemistry, pharmacology and toxicology of bolesatine is discussed in this article.
- Klíčová slova
- Rubroboletus,
- MeSH
- Agaricales * chemie klasifikace MeSH
- antitumorózní látky terapeutické užití MeSH
- fungální proteiny * chemie izolace a purifikace toxicita MeSH
- gastroenteritida chemicky indukované komplikace terapie MeSH
- glykoproteiny chemie otrava toxicita MeSH
- houby chemie klasifikace MeSH
- lektiny chemie imunologie MeSH
- lidé MeSH
- mykotoxiny * chemie otrava toxicita MeSH
- otrava houbami klasifikace komplikace terapie MeSH
- proteosyntéza genetika imunologie účinky léků MeSH
- ricin chemie otrava toxicita MeSH
- statistika jako téma MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
- přehledy MeSH
Galectin-3 (Gal-3) is recognized as a prognostic marker in several cancer types. Its involvement in tumor development and proliferation makes this lectin a promising target for early cancer diagnosis and anti-cancer therapies. Gal-3 recognizes poly-N-acetyllactosamine (LacNAc)-based carbohydrate motifs of glycoproteins and glycolipids with a high specificity for internal LacNAc epitopes. This study analyzes the mode and kinetics of binding of Gal-3 to a series of multivalent neo-glycoproteins presenting complex poly-LacNAc-based oligosaccharide ligands on a scaffold of bovine serum albumin. These neo-glycoproteins rank among the strongest Gal-3 ligands reported, with Kd reaching sub-nanomolar values as determined by surface plasmon resonance. Significant differences in the binding kinetics were observed within the ligand series, showing the tetrasaccharide capped with N,N'-diacetyllactosamine (LacdiNAc) as the strongest ligand of Gal-3 in this study. A molecular model of the Gal-3 carbohydrate recognition domain with docked oligosaccharide ligands is presented that shows the relations in the binding site at the molecular level. The neo-glycoproteins presented herein may be applied for selective recognition of Gal-3 both on the cell surface and in blood serum.
- MeSH
- galektin 3 chemie metabolismus MeSH
- glykoproteiny chemie farmakologie MeSH
- laktosa analogy a deriváty chemie MeSH
- lidé MeSH
- ligandy MeSH
- sérový albumin hovězí chemie MeSH
- simulace molekulového dockingu * MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Human milk is a source of glycoconjugates, sialylated forms of which enrich the newborn immature immune system and are crucial for their proper development and well-being. Here, we analyzed the expression of α2,3-/α2,6-sialylated glycotopes on skim milk glycoproteins over lactation. Milk samples were analyzed by lectin-blotting using α2,3- and α2,6- sialic acid specific Maackia amurensis (MAA) and Sambucus nigra (SNA) lectins and sialyl- and asialyl-T antigen specific Artocarpus integrifolia (Jacalin) and Arachis hypogaea (PNA) lectins. The reactivities of MAA, SNA, Jacalin and PNA with milk glycoproteins showed that they are heavily decorated with α2,3-/α2,6-linked sialic acid and sialyl-T antigen and to a lesser degree with asialyl-T antigen. Despite individual differences of particular glycoproteins, a sharp and significant decline of α2,6-sialylated glycotopes and sialyl-T antigens and a weaker but significant decrease of α2,3-sialylated glycotopes and asialyl-T antigens on milk glycoproteins during milk maturation was observed. The expression of α2,3-/α2,6-sialylated glycotopes, sialyl- and asialyl-T antigens corresponds to milk maturation but differs in relation to the analyzed glycoprotein. Sialylated milk glycoproteins are considered as a part of innate immunity provided to neonates. Further investigations are needed to understand if they may be useful in milk banking to control the biochemical quality of milk.
- Klíčová slova
- sialyl-T antigen,
- MeSH
- antigeny povrchové MeSH
- časové faktory MeSH
- chemické techniky analytické MeSH
- glykoproteiny * chemie MeSH
- kyseliny sialové analýza chemie MeSH
- laktace MeSH
- lektiny chemie MeSH
- lidé MeSH
- mateřské mléko * chemie MeSH
- muciny * chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
Galectin-3 (Gal-3), a member of the β-galactoside-binding lectin family, is a tumor biomarker and involved in tumor angiogenesis and metastasis. Gal-3 is therefore considered as a promising target for early cancer diagnosis and anticancer therapy. We here present the synthesis of a library of tailored multivalent neo-glycoproteins and evaluate their Gal-3 binding properties. By the combinatorial use of glycosyltransferases and chemo-enzymatic reactions, we first synthesized a set of N-acetyllactosamine (Galβ1,4GlcNAc; LacNAc type 2)-based oligosaccharides featuring five different terminating glycosylation epitopes, respectively. Neo-glycosylation of bovine serum albumin (BSA) was accomplished by dialkyl squarate coupling to lysine residues resulting in a library of defined multivalent neo-glycoproteins. Solid-phase binding assays with immobilized neo-glycoproteins revealed distinct affinity and specificity of the multivalent glycan epitopes for Gal-3 binding. In particular, neo-glycoproteins decorated with N',N″-diacetyllactosamine (GalNAcβ1,4GlcNAc; LacdiNAc) epitopes showed high selectivity and were demonstrated to capture Gal-3 from human serum with high affinity. Furthermore, neo-glycoproteins with terminal biotinylated LacNAc glycan motif could be utilized as Gal-3 detection agents in a sandwich enzyme-linked immunosorbent assay format. We conclude that, in contrast to antibody-based capture steps, the presented neo-glycoproteins are highly useful to detect functionally intact Gal-3 with high selectivity and avidity. We further gain novel insights into the binding affinity of Gal-3 using tailored multivalent neo-glycoproteins, which have the potential for an application in the context of cancer-related biomedical research.
- MeSH
- aminocukry chemická syntéza chemie metabolismus MeSH
- galektin 3 antagonisté a inhibitory metabolismus MeSH
- glykoproteiny chemická syntéza chemie metabolismus farmakologie MeSH
- glykosylace MeSH
- lidé MeSH
- ligandy MeSH
- oligosacharidy chemická syntéza chemie metabolismus MeSH
- sérový albumin hovězí chemická syntéza chemie metabolismus farmakologie MeSH
- skot MeSH
- techniky kombinatorické chemie MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Carbohydrates constitute a structurally and functionally diverse group of biological molecules and macromolecules. In cells they are involved in, e.g., energy storage, signaling, and cell-cell recognition. All of these phenomena take place in atomistic scales, thus atomistic simulation would be the method of choice to explore how carbohydrates function. However, the progress in the field is limited by the lack of appropriate tools for preparing carbohydrate structures and related topology files for the simulation models. Here we present tools that fill this gap. Applications where the tools discussed in this paper are particularly useful include, among others, the preparation of structures for glycolipids, nanocellulose, and glycans linked to glycoproteins. The molecular structures and simulation files generated by the tools are compatible with GROMACS.
We introduce an optimized periodic acid-Schiff´s staining of glycoproteins on 1D electrophoretic gels. Thanks to heating in a household microwave oven the protocol of standard periodic acid-Schiff´s staining has been accelerated from 6 h to below 10 min employing standard chemistry. At the same time, we show that the microwave-assisted glycoprotein staining is at least as sensitive as the conventional approach. All glycoproteins stained by the microwave-accelerated procedure were successfully identified using MALDI TOF/TOF mass spectrometry. The ensuing reduction in gel staining time and simplification of the staining protocol should significantly increase laboratory throughput when glycoprotein detection on electrophoretic gels is required in large numbers.
- MeSH
- barvení a značení metody MeSH
- barvicí látky chemie MeSH
- časové faktory MeSH
- elektroforéza v polyakrylamidovém gelu metody MeSH
- glykoproteiny analýza chemie MeSH
- kyselina jodistá chemie MeSH
- mikrovlny * MeSH
- reprodukovatelnost výsledků MeSH
- rosanilinová barviva chemie MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
