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Expression and binding properties of human galectin-4
Friede, Tereza ; Bojarová, Pavla (advisor) ; Kavan, Daniel (referee)
Galectin-4, a member of the galectin family known for its affinity to β-galactosides, belongs to tandem-repeat galectins and consists of two distinct subunits connected with a peptide linker. Galectins play a key role in various biological processes and are involved in the pathogenesis of many diseases. Colorectal cancer (CRC) tissue shows a lower expression of galectin-4 compared to healthy colon tissues, and the lower expression of galectin-4 promotes tumor progression and transport. Galectin-4 was found to inhibit tumorigenesis of CRC cells via Wnt/β-catenin and IL-6/NF-κB/STAT3 signaling pathways, which play a key role in CRC development. In inflammatory bowel diseases such as Crohn's disease and ulcerative colitis, galectin-4 interacts with CD4+ T lymphocytes, influencing their behavior in the immunological synapsis. Furthermore, galectin-4, primarily expressed in the gastrointestinal tract, participates in the apical transport in epithelial cells, promotes the healing of intestinal epithelial wounds, participates in the adaptive immune response and has multiple roles in the central nervous system. Studying inhibitors of galectin-4 helps to understand its specific roles in these processes, and thus to identify new potential therapeutic targets. In this work human galectin-4 was heterologously...
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Production of human milk oligosaccharides in the cell factory of E. coli
Havrdová, Jana ; Bojarová, Pavla (advisor) ; Smrček, Stanislav (referee)
Human milk oligosaccharides (HMOs) are among the most abundant components of human breast milk and are essential for the development of neonatal health. It is very challenging to isolate these oligosaccharides from animal milk, especially the less abundant ones. Therefore, different approaches are to be sought after. Chemical and enzymatic syntheses of these compounds are labor-intensive, and expensive affording low yields. A newly adopted approach to HMO synthesis is through bac- terial cell factories, in which genetically engineered bacterial strains can use cheap carbohydrate substrates and convert them into specific oligosaccharides. The aim of this thesis is to examine the feasibility of using selected bacterial strain (E. coli) for the production of HMOs. With lactose as a glycan substrate, the bacterial host has to be β-galactosidase deficient, otherwise, the substrate would be degraded. In order to generate higher lactose intake in the cell, a crp gene that encodes for the positive transcriptional regulator (Catabolite Activator Protein - CAP) can be incorporated into the host organism. The cloned plasmid - pRSFDuet- 1-crp was used for the transformation of into the selected bacterial strain. Lactose from the cells was purified by gel chromatography and the influence of CAP over expression...
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Production and characterization of novel aryl sulfotransferases and their use in bioanalytics
Petránková, Barbora ; Bojarová, Pavla (advisor) ; Křížek, Tomáš (referee)
Polyphenols are the most common natural substances found in food. The largest group is represented by flavonoids, phenolic acids and catechols. Polyphenols are metabolized in the body during the biotransformation phase II, they can be sulfated, glucuronylated or methylated. To study biotransformation, it is necessary to have defined standards. Sulfated polyphenols can be prepared by enzymatic sulfation, which needs much milder reaction conditions compared to chemical sulfation. Prokaryotic sulfotransferases use 3'-phosphoadenosine-5'-phosphosulfate (PAPS) as a sulfate donor for sulfation, which is very expensive and difficult to regenerate. An alternative is the group of bacterial aryl sulfotransferases (EC 2.8.2.22), which catalyze the transfer of a sulfate group from a phenolic donor to a phenolic acceptor. Two bacterial aryl sulfotransferases from Desulfitobacterium hafniense and Escherichia coli have been described in the literature so far. The present thesis deals with the production, purification and biochemical characterization of two new bacterial aryl-sulfotransferases from Desulfosporosinus sp. HMP52 and Salmonella bongori. The enzymes were heterologously expressed in E. coli and purified by affinity chromatography. Their pH and temperature optimum, stability in organic solvents and...
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Rutinosidase from Aspergillus niger - analysis of active site and its mutagenesis
Šidáková, Anna ; Bojarová, Pavla (advisor) ; Palyzová, Andrea (referee)
Rutinosidases (α-L-rhamnosyl-β-D-glucosidases) from Aspergillus niger (AnRut) are glycosidases (EC 3.2.1) that catalyze the hydrolysis of the glycosidic bond between the aglycone and the disaccharide residue rutinose. The dual substrate specificity of this enzyme group describes the parallel activity towards the substrates rutin (carrying a rutinosyl disaccharide residue) and isoquercitrin (carrying a glucosyl residue). The active site of AnRut is more complex than that of other glycosidases and is composed of the catalytic amino acids Glu210 and Glu319 in the active-site cleft and a side tunnel. This untraditional structure with distinct interactions in the tunnel and active-site cleft is the probable reason for the enzyme exceptional substrate specificity. Through point or multiple mutations of the enzyme, we can modify its primary and secondary structure, thus causing a significant shift in substrate specificity. The main goal of this thesis is the analysis of three distinct mutant variants of AnRut rutinosidase; their production, purification, and the study of the influence of the mutations on the substrate specificity of the enzymes. All variants were designed based on molecular modeling. The substrate specificity was determined by reactions of the mutant variants with previously unstudied...
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In vivo biotinylation as a new method of galectin labeling
Balogová, Soňa ; Bojarová, Pavla (advisor) ; Kavan, Daniel (referee)
Galectins are animal lectins with affinity to terminal β-galactosides. These lectins are characterized by an evolutionarily conserved carbohydrate recognition domain (CRD). Galectins are involved in many processes in vivo, such as cell adhesion; signaling; cell proliferation and carcinogenesis. They can be used as markers in certain types of cancer and also for the targeted binding of therapeutics in the diagnosis and therapy of cancer. Galectins have been defined as a protein family composed of 15 members (11 of which are known in humans). They are classified into three subgroups, according to their structure: prototypical; chimeric; tandem-repeat type. Galectin-8 belongs to tandem-repeat galectins, which consist of two different subunits connected with a peptide linker; they contain two different CRD domains, and are therefore bivalent. Galectin-glycan interactions are based on the affinity of galectin CRDs to glycans containing a terminal β-galactoside, and this affinity is formed by the set of conserved amino acids within each CRD. These interactions can be investigated by many methods, such as X-ray crystallography; competitive ELISA; biolayer interferometry. Due to the extensive involvement of galectins in biological processes, there are many methods of labeling galectins for their detection....
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Glycocalixarenes and their affinity to human galectins
Bálková, Denisa ; Bojarová, Pavla (advisor) ; Kavan, Daniel (referee)
Calix[n]arenes are macrocyclic compounds synthesized by the oligomerisation of phenol and formaldehyde. They can have different numbers of phenol rings and can adopt various conformations. As they can be easily modified, they are widely used in many ways, especially in industry or medicine. Modifications can comprise many functional groups such as hydroxyl, amine or carboxyl. Moreover, they can be functionalized with carbohydrates. In such a case, glycocalyx[n]arenes are formed. Galectins, S-type lectins, are capable of specific recognition and subsequent binding of a number of glycoconjugates. However, it is possible only when the molecule presents β-D-galactosides. Galectins have different binding affinities to particular ligands. It has been proven that if the β-D-galactosides are conjugated to a multivalent carrier, galectins have higher affinity to the resulting glycoconjugate. With these ligands it is possible to affect many functions of galectins such as cell adhesion, regulation of apoptosis, proliferation, cell signalling pathways and immune response. Through these pathways, galectins can also cause serious diseases such as autoimmune diseases or carcinogenesis. Thus, the goal of many studies is to find such galectin ligands that could prevent these disorders or help with their treatment....
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Mutant glycosidases with a high substrate specificity and their analysis
Nekvasilová, Pavlína ; Bojarová, Pavla (advisor) ; Lichá, Irena (referee)
β-N-acetylhexosaminidases (EC 3.2.1.52, GH 20) are retaining exo-glycosidases that in vivo cleavage both β-N-acetylglucosamine (GlcNAc) or β-N-acetylgalactosamine (GalNAc) residues fom glycostructures. Under suitable reaction conditions, these enzymes are able to synthesize the glycosidic bond in good yields. Substitution of selected amino acid(s) in the emzyme active site by site-directed mutagenesis may change the enzyme's substrate specificity or suppress the hydrolytic activity of the enzyme in favor of synthesis. The present thesis deals with three mutant β-N-acetylhexosaminidases from Talaromyces flavus, in which the amino acid residues responsible for binding to C-4 hydroxyl of the substrate (Arg218, Glu546) were exchanged for amino acids proposed on the basis of molecular modeling. The effect of introduced single point mutations on substrate specificity of prepared enzymes was studied. Mutant β-N-acetylhexosaminidases were heterologously expressed in Pichia pastoris and characterized. Furthermore, transglycosylation reactions with these enzymes were performed. The prepared carbohydrate products were characterized by NMR.
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Enzymatic fucosylation of oligosaccharides
Horáčková, Karolína ; Bojarová, Pavla (advisor) ; Moserová, Michaela (referee)
Fucosylated oligosaccharides occur naturally in the mammalian body, for example in the form of blood group antigens or human milk oligosaccharides. Human milk oligosaccharides act as inhibitors of pathogen binding to infantile gastrointestinal epithelial cells, thereby preventing diarrheal diseases. The most abundant human milk oligosaccharides are 2' fucosylgalactose and 2' fucosyllactose using mutant α α 2' Key words carbohydrate; enzymatic synthesis; fucosidase; fucosylation; human milk
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Analysis of sulfated flavonoids by HPLC-MS/MS
Petránková, Barbora ; Bojarová, Pavla (advisor) ; Kozlík, Petr (referee)
The aim of this work is to find the optimal separation conditions in high performance liquid chromatography for the analysis of sulfated polyphenolic substances, especially flavonoids, phenolic acids and catechols. Three chromatographic columns were used in the development of the method. ZIC-HILIC column with polar organic phase and C18 and Kinetex PFP columns with reverse phase. We focused in particular on a short time of separation, sharp tailless peaks and separation of all components of the mixture. Using a Kinetex PFP column, the optimal method for separating the test mixtures was found. The use of a mobile phase of 10 mM ammonium acetate/0,1% fromic acid and methanol was shown to be the most appropriate, with a mobile phase flow rate of 0,6 ml/min. By modifying the gradient and temperature, all components of the tested mixtures were separated, the peaks were sharp, and the separation time in most cases ranged do not exceed 10 min. The method is also directly applicable in conjunction with a mass detector. Keywords: High Performance Liquid Chromatography (HPLC), flavonoids, polyphenolic substances, sulfation, separation
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