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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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Expression and purification of proteins with unnatural aminoacids, and determination of their structure by combination of chemical or photochemical modification and mass spectrometry
Bortel, Tomáš ; Ječmen, Tomáš (advisor) ; Kavan, Daniel (referee)
Residue-specific non-natural amino acid (NNAA) incorporation has become a widely used approach to introduce bioorthogonal groups and therefore functionalities into proteins26 . These functionalities are harnessed through click chemistry, conjugating labelled proteins to affinity or fluorescent probes. A less utilized approach involves probing the effects of bioorthogonal groups on the structure-function relationship of labelled proteins47 . In order to investigate these relationships, there is a need to express large amounts of proteins with maximal incorporation of NNAAs. Here, we employed photo-methionine (pMet), azidohomoalanine (AHA) and homopropargylglycine (HPG) as methionine (Met) surrogates. We investigated the impact of these NNAAs on bacterial growth of prototrophic E. coli BL21 or Met- -auxotrophic E. coli B834 in Met-free MM-M9 medium. We monitored the expression of cytochrome b5 (cyt b5) and MBP-GFP. Using MS and LC-MS based approaches, we determined the NNAA incorporation in these recombinant proteins. Prototrophic E. coli BL21 expressed significantly higher amounts of cyt b5 compared to B834 with pMet and AHA, but the incorporation rates fell sharply after 4 hours. In contrast, Met-auxotrophic B834 expressed smaller amounts of protein, but with incorporation of pMet in 50 - 70% range...
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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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Expression and purification of N-terminal fragment of filamentous hemagglutinin from Bordetella Pertussis in E. Coli
Jurnečka, David ; Stiborová, Marie (advisor) ; Kavan, Daniel (referee)
: Whooping cough is highly contagious disease caused by gram-negative bacteria Bordetella pertussis. During infection the bacteria produces many types of toxins and adhesive molecules including a filamentous hemagglutinin(FHA). FHA is 220 kDa surface-exposed and secreted protein, which plays a key role in host-cell interactions. The project aims at construction of heterologous expression system for production of the N-terminal part of B. pertussis FHA (FHA1-862) in E. coli. The expression vector is composed of system of two independent T7/Lac promoters and enables secretion of FHA1-862 into the culture media. Downstream of the first promoter is fhaB gene encoding FHA1-862 and the letter is followed by fhaC gen encoding the FhaC transport protein, which allows translocation of FHA from periplasmic space to extracellular milieu. FHA1-862 was successfully secreted in E. coli strain BL21 carrying plasmid pMM100 (Laclq) at 30 řC and purified by affinity chromatography on Cellufine resin. These results indicate that FHA1-862 protein can be produced in E. coli, however, the system is inefficient and the yield of the protein is very low. (In Czech)
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Monitoring of leukemic cell line amino acid metabolism changes after Quambalarine B treatement
Matoušková, Zuzana ; Kavan, Daniel (advisor) ; Prošková, Veronika (referee)
Leukemia is the most common cancer of children, moreover it is also not uncommon of elderly patients. Research has focused on the development of specific antileukemic drugs in recent years. Abnormalities in tumor cell metabolism that can be targeted during treatment appear to be the key. Natural 1,4-naphthoquinones, including quambalarin B produced as a secondary metabolite by the basidiomycetes of Quambalaria cyanescens, are known for their therapeutic effects. Not surprisingly, Quambalarine B has also been shown to inhibit cell proliferation in some leukemic cell lines and subsequently caused cell death. In the present thesis, I tried to observe changes in amino acid metabolism by monitoring amino acid levels in the intracellular and extracellular environment of leukemic cells after treatment with Quambalarine B using amino acid analysis with fluorescence detection. The observation was performed in Jurkat, Ramos and THP-1 cell lines, each of these lines represents another type of leukemic disease. [IN CZECH] Key words Amino acid analysis, amino acid metabolism, Quambalarine B, leukemia
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The novel combinations of experimental approaches: mass spectrometry (MS) and photo-induced surface labelling, electron release (PIER), or cross-linking (PIXL)
Tuzhilkin, Roman ; Šulc, Miroslav (advisor) ; Kavan, Daniel (referee)
Countless electron transport/transfer (ET) processes occur in living organisms every day. Therefore, their study is a crucial field of modern structural and functional proteomics. In many cases model proteins like azurin from P. aeruginosa are utilised in experiments. This blue copper protein is favoured due to a characteristic absorbance maximum at 630 nm in Cu(II) redox state of the central Cu atom. During its oxidation to Cu(I) state the A630 value decreases allowing UV-Vis detection of ET reaction progress. We have introduced a structural photoinducible analogue of canonical amino acid Met - L-2-amino-5,5-azihexanoic acid (photo-Met) - into azurin structure to study oligomerization in solution via photo-induced cross-linking (PIXL). Using previously optimised protocols for recombinant expression in E. coli B834 we have inserted photo-Met into azurin moieties: wild type azurin and Az2W mutant where two adjacent W residues with confirmed role in electron hopping across protein-protein interface are present. The incorporation percentage of photo-Met in analysed samples was determined after SDS-PAGE and in-gel protease digestion via MALDI-TOF MS. PIXL was employed to study azurin-azurin interaction and oligomerization under different total concentrations of protein (in range of 15-300 µM). The...
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Studies on DCL-1, receptor of dendritic cells, using NMR techniques
Pospíšilová, Eliška ; Kavan, Daniel (advisor) ; Schneider, Bohdan (referee)
(EN) The DCL-1 receptor (CD302) is predominantly expressed on the surface of dendritic cells and according to its sequence similarity DCL-1 is classified as a C-type lectin. Since its extracellular domain lacks single motives for carbohydrate binding in coordination with calcium ions, it is probable that the process of carbohydrate binding does not occur through the classical pathway as described in the case of mannose receptor or the DEC-205 receptor. Due to its colocalization with F-actin there is a presumption, that DCL-1 plays a role in cell adhesion and migration. Another role of DCL-1 could be the participation in endocytosis and subsequent targeting to lysosomes. DCL-1 was also put in connections with various pathologies in last few years Experiments described in this work can be divided into three sections. In the first part I dealt with production of a protein construct based on the extracellular domain of DCL-1. The protein was produced in M9 minimal medium with the only source of nitrogen 15 NH4Cl and the only source of carbon 13 C glucose. The result was 15 N,13 C labelled protein, used for NMR measurements. The second part is dedicated to the analysis of NMR spectra, which enabled us to assign frequencies of protein backbone and aliphatic side chains atoms. On the base of the backbone...
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Multivalent carbohydrate ligands of galectins
Hovorková, Michaela ; Křen, Vladimír (advisor) ; Kavan, Daniel (referee)
Galectins are proteins, wich belong to a group of lectins that are able to bind to saccharide units and they specifically recognize glycans exposed to the surface of the cells. Galectins participate in vivo, for example, in carcinogenesis, angiogenesis or fibrosis. Their occurrence increases significantly in connection with a number of pathogenic processes, therefore they can be used as markers for some types of cancer or cardiopathology and also for the targeted binding of therapeutics and/ or imaging agents in diagnosis and therapy. Galectin-3 has a specific structure known as chimeric and it is capable of forming multivalent oligomers. The natural ligands of galectins are glycans containing terminal β-galactosides, especially N-acetyllactosamine, but the binding of monovalent glycans is very weak. Glycoconjugates with high affinity to galectin receptors are optimally multivalent, biocompatible and stable in vivo. These criteria accomplish carbohydrate ligands conjugated to soluble and structurally flexible N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers. In this work two types of functionalized disaccharides based on N-acetyllactosamine (Galβ4GlcNAc) and its structural analogue of N,N'-diacetyllactosamine (GalNAcβ4GlcNAc) was prepared by enzymatic synthesis. For the synthesis were used...
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