Lectins are tissue specific carbohydrate binding proteins with possible functions in invertebrate immunity and pathogen transmission. The main goal of this study was to identify hemolymph lectins in three different tick species. Three proteins with molecular weights of 58 kDa, 75 kDa and 180 kDa were detected in all investigated species using antibodies directed against hemagglutination activity of Rhipicephalus appendiculatus hemolymph. These proteins were characterized by biochemical methods such as Schiff/periodate staining, lectin blotting, enzymatic deglycosylation, hemagglutination analysis, immunoblotting, and mass spectrometry.

Dipeptidyl peptidase IV (DPP-IV) is a serine protease, which executes its proteolytic activity by cleaving X-Pro dipeptides from the N-termini of its substrates. Furthermore, DPP-IV exhibits many biological functions independent of its enzymatic aktivity. Previous studies in our laboratory proved increased expression of DPP-IV in high-grade astrocytic tumours. To evaluate the enzymatic and non-enzymatic functions of DPP-IV in a glioma model, clones of asctrocytic cell line U373MG transfected by enzymatically inactive, mutated DPP-IV (mutDPP-IV) and enzymatically active, wild type DPP-IV (wtDPP-IV), were prepared. Enzymatically inactive mutDPP-IV was prepared using point mutation the active site serine residue. Cells U373MG were transfected using a doxycycline inducible Tet-On® system. For further analysis of the transgenic forms of DPP-IV, methods were used for verification of protein expression, enzymatic activity and subcellular localization. Doxycycline induced U373MG mutDPP-IV and U373MG wtDPP-IV cells, expressing mutated and wild type DPP-IV, respectivelly, exhibited increased expression of transgenic DPP-IV in a concentration and time dependent manner. Doxycycline induced U373MG wtDPP-IV cells exhibited both increased expression and enzymatic activity of DPP-IV. In contrast, DPP-IV enzymatic...

Carbohydrates have an essentials role in wide range of biological phenomena. It is well known that most of the eukaryotic proteins are glycosylated and that their glycosylation undergoes dynamic changes, nevertheless the biological imperative for these modifications is still not fully understood. However, one area in which the importace of cell surface glycosylation has recently been the subject of active investigations is the tumor plasma membrane biology, where many changes in glycosylation have been found useful for diagnosis, and mostly recent, even for the therapies of malignant disease. Interestingly cell surface glycoconjugates, namely N-linked and O-linked oligosaccharides have been found therapeutically attractive for treatment of certain tumors. And although our understanding of the participation of these principal glycan classes in tumorigenesis is far from complete, there are already several examples of carbohydrate-based antitumor vaccines. Therefore, we decided to give this issue more attention, especially the molecular mechanisms responsible for identifying changes in glycosylation of the surface of tumor cells of the immune system. Although in the past in our laboratory identified a receptor-type lectin specific lectin receptors on natural killer cells, very little is yet known...

NK cells are important part of immune system, recognizing and eliminating tumor cells and cells infected by viruses. For the target cell recognition, binding of ligands by activating receptors plays a crucial role. Activating receptor NKp30, protein of family of natural cytotoxicity receptors, is able to bind multiple ligands either present on tumor cell surface or being part of some viruses. B7-H6 is one of the ligands of NKp30 and its specific constitutive expression on some tumor cells and cell lines makes it an interesting biological target. Although the NKp30/B7-H6 complex structure has been solved, structural basis of some important features of their binding is not explained yet. Soluble form of NKp30 receptor binding domain creates oligomers, presence of which is dependent on C-terminus length of its domain and its N-glycosylation; however, structural insight into formation of the oligomers and their significance is not known. Furthermore, binding affinity of NKp30 to its ligands is dependent on presence of its glycosylation and glycosylation type. We have already found out that NKp30 oligomerization is dependent on its glycosylation. In my work, I attempted to gain detailed functional and structural information about oligomerization of NKp30 and its binding to B7-H6 by multimethodical...

Natural killer (NK) cells are a subpopulation of effector lymphocytes with cytotoxic activity and cytokine-producing functions considered as an integral part of the innate immune response. Functions of NK cells include tumour elimination, engagement and regulation of antiviral immune responses and regulation of immune cells by production and secretion of chemokines and cytokines. CD69 is a C-type lectin-like transmembrane receptor expressed in NK cells. CD69 is an activating receptor and acts also as a very early marker of lymphocyte activation. Putative protein ligands have been described for CD69 in the last years: Galectin-1, S1P1, S100A8/S100A9 and Myl9/12. Galectin-1 is a prototypical lectin characterized by the presence of a common lectin structural fold and a carbohydrate recognition domain involved in carbohydrate binding. Galectin-1 was identified as a binding partner for CD69 based on biological and functional studies, but structural details about the complex are still missing. This thesis describes the successful establishment of an expression protocol for a tag-less cysteine-less mutant of galectin-1 and the study of the interaction between galectin-1 and NK cell receptors. The interaction was studied using microscale thermophoresis and confirmed as dependent on the presence of a...

Tick-borne encephalitis virus (strain Hypr) was serially subcultured in PS cells and tick cell line IRE/CTVM19, producing four different viral variants. Biological properties of these new variants were investigated in mouse model. Possible determinants of virulence were found by full-genome sequencing. The role of glycosylation for tick-borne encephalitis virus was evaluated.

The aim of the study is the purification and characterization of hemelipoglycoprotein, a carrier protein from the ornate sheep tick Dermacentor marginatus. The protein was characterized by biochemical methods with emphasis on its glycosylation and native molecular weight, and examining its carbohydrate-binding specificity.

Tato práce se zabývá proteomickými studiemi ječmene v souvislosti s výrobou piva. Ječmen patří mezi nejvýznamnější plodiny na světě a je využíván hlavně pro sladovnické účely, nejčastěji pro pivovarnictví. Studium proteinů ječmene během sladování a výroby piva poskytuje informace o změnách v proteinovém složení nebo jejich posttranslačních modifikacích. Jelikož jsou proteiny v ječmeni a jejich změny zásadní pro kvalitu sladu a piva, proteomické studie ječmene mají potenciál pro zlepšení procesu sladování a pivovarnictví. Hlavním cílem této práce je studium ve vodě rozpustných proteinů ječmene a jejich změn, ke kterým dochází během sladování a výroby piva. Rozdíly v proteinovém složení byly sledovány pomocí gelové elektroforézy, kapalinové chromatografie na reverzní fázi, gelové chromatografie a MALDI-TOF hmotnostní spektrometrie. Během sladování se vlivem klíčení zrna zvyšuje množství některých proteinů a také jsou tvořeny nové proteiny. V průběhu vaření piva se naopak v důsledku vysoké teploty a enzymatické aktivity proteáz mnoho proteinů rozkládá. Těmto drsným podmínkám odolají jen některé proteiny, které přechází až do piva a mohou ovlivnit jeho kvalitu. Dále byly zkoumány různé odrůdy ječmene a jejich rozdíly. Byly porovnány odrůdy povolené pro výrobu certifikovaného Českého piva s jednou osvědčenou sladovnickou odrůdou a jednou nesladovnickou odrůdou ječmene. Kromě toho byly studovány v alkoholu rozpustné proteiny ječmene a jejich změny v průběhu sladování. Zvláštní pozornost byla věnována vybrané skupině posttranslačních modifikací proteinů: glykosylacím. Neenzymaticky glykosylované proteiny ječmene (neboli glykované proteiny) jsou tvořeny v průběhu sladování kvůli přítomnosti velkého množství glukózy uvolněné z rozkladu škrobu. Glykované proteiny ovlivňují stabilitu proteinů a kvalitu piva, obzvlášť pěnotvorný účinek. Enzymatické N-glykosylace představují nejčastěji studované posttranslační modifikace u rostlin, protože glykoproteiny hrají klíčovou roli v různých biologických funkcích. Glykoproteiny jsou často přítomny v malém množství, a proto je pro jejich analýzu potřebné obohacení glykoproteinů z komplexní směsi. Pro studium glykoproteinů byla využita afinitní chromatografie s lektinem concanavalin A. Kromě toho byla také optimalizována analýza sacharidové části glykoproteinů. Tato disertační práce přináší důležité informace o proteinech ječmene, jejich změnách a analýze, které budou užitečné pro další studium.

Congenital disorders of glycosylation (CDG) represent a rapidly growing group of rare inherited metabolic diseases with estimated prevalence as high as 1:20 000, which are caused by genetic defects that impair the process of glycosylation, i.e. the enzymatic addition of a specific saccharide structure onto a protein or lipid backbone. Due to non-specificity and variability of clinical symptoms in the patients, the medical diagnosis of CDG remains extremely challenging and significantly relies on accurate biochemical and genetic analyses. The overall goal of the present dissertation thesis was to study CDG at the biochemical and molecular genetic level in the context of the Czech and Slovak Republic, which involved three specific aims: A.) to introduce and optimize laboratory screening methods for CDG detection in a group of clinically suspected patients, B.) to determine the corresponding genetic defect in the positive patients selected via CDG screening and to study the pathobiochemical aspects of specific CDG types at the cellular level, and C.) to analyze glycosylation disturbances of non- CDG etiology. Contributions of this work include optimization of isoelectric focusing of apolipoprotein C-III (ApoC-III) as a screening method for O-glycosylation abnormalities, as well as the description of...

Dolichol is a membrane lipid, which carries monnosaccharides and glycans for N-linked protein glycosylation and glycosylphosphatidylinositol-anchor biosynthesis occuring in endoplasmic reticulum. Its structure is composed of isoprenoid units. Dolichol is present in all tissues and in most of the membrane organelles of eukaryotic cells. Recently some types of congenital disorders of glycosylation have been described as a consequence of dolichol biosynthesis and metabolism defects, which are not detectable by standard methods. The aim of this diploma thesis was to analyze dolichol content in urine and in different tissues from patients with deficiency in dolichol biosynthesis by mass spectrometry and to study the impact of these defects on energetic metabolism. Biological material for this study consisted of urine samples from 76 controls with age ranging from 1 months to 81 years, 6 patients with congenital disorders of glycosylation and 43 patients with suspicion of congenital disorder of glycosylation; samples of frontal cortex, liver, muscle and heart tissues from 2 patients with mutation in NUS1 gene and controls. Urine samples were stored at -20 řC and tissue homogenates were stored in -80 řC until analysis. Lipid fraction after extraction was separated by liquid chromatography. Dolichols were...