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Characterization of cystatin in the tick \kur{Ixodes ricinus}
PERNER, Jan
In numerous organisms, proteases and their proteinaceous inhibitors constitute a major defence mechanism. As vectors are in constant touch with pathogens their proteolytic activity is of crucial importance. In Ixodes ricinus (IR), the main vector of Lyme disease in Europe, the midgut cells process bloodmeal particles as well as provide a major barrier to a pathogen invasion. Tick salivary secretion plays a indispensable role in both bloodmeal completion and inadvertant pathogen transmission. This thesis describes 3 IRcystatins, cysteine protease inhibitors, using standard biochemical and molecular protocols. Moreover, this work provides evidence that cystatin 3 is likely to be involved in gut processes. This study also highlights recent knowledge on tick physiology during blood-feeding.
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Prolyl endopeptidase from the tick Ixodes ricinus
Petrvalská, Olívia ; Konvalinka, Jan (advisor) ; Ryšlavá, Helena (referee)
The ticks are important blood-feeding parasites and vectors of pathogens. The hard tick Ixodes ricinus is the most common species in the Czech Republic that transmits Lyme disease and tick-borne encephalitis. Proteases of the ticks are potential drug targets for the development of new vaccines against these parasites. This work is focused on biochemical analysis of a prolyl endopeptidase from I. ricinus, which has not been studied so far. The prolyl endopeptidase was identified in the extract from the tick gut tissue by the measurement of enzyme activity and by visualization on SDS-PAGE after labelling with activity-based probe. The tick prolyl endopeptidase is probably involved in the proteolytic digestion of host blood proteins based on the highest specific activity found in the gut tissue and its upregulation during the blood-feeding period. Biochemical analysis showed that the enzymatic activity of prolyl endopeptidase is (1) dependent on a free cysteine residue in a close proximity of the active site, (2) optimal at a pH range between 8 and 9, and (3) selectively inhibited by peptide inhibitors Z-Ala-Pro-CMK and Z-Pro-Pro-CHO. Key words: prolyl endopeptidase, proteolysis, enzyme activity, substrate specificity, tick (In Czech)
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Cathepsin L from the hard tick Ixodes ricinus: analysis of proteolytic activity and its regulation
Talacko, Pavel ; Konvalinka, Jan (advisor) ; Ryšlavá, Helena (referee)
The hard tick Ixodes ricinus is an important blood-feeding parasite that transmits tick- borne diseases, such as tick-borne encephalitis and Lyme disease. Ticks employ a battery of proteolytic enzymes, including cathepsins, to digest their bloodmeal. These proteins are potential targets for the development of anti-tick vaccines. This work is focused on cathepsin L from I. ricinus (IrCL), namely its isoenzymes IrCL1 and IrCL3. IrCL1 was expressed in Pichia pastoris and chromatographically purified. Its substrate specifity was determined by the cleavage of (a) peptide fluorogenic substrates and (b) protein substrates analyzed by mass spectrometry. The proteolytic activity of IrCL1 was modulated by its interaction with glycosaminoglycans, which affected the pH optimum value. Futhermore, a proteolytically active mutant of IrCL1 with reduced number of N-glycosylation sites was prepared; this form will be used for crystallization experiments. IrCL3 was expressed in Escherichia coli, refolded and activated to its active form. The proteolytic activity of IrCL3 is in many ascpects similar to that of IrCL1, including substrate specifity, acidic pH optimum and activity modulation by glycosaminoglycans. Key words: cysteine proteases, cathepsin L, hard tick I. ricinus, substrate specifity, proteolytic activity...
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Ricinusin {--} a new antimicrobial peptide isolated from the hard tick \kur{Ixodes ricinus}: the third member of the tick histidine-rich defense protein family.
DORŇÁKOVÁ, Veronika
New gene encoding antimicrobial protein (ricinusin) was isolated from I. ricinus. The 405 nt cDNA fragment contained a 135 aa ORF, encoding a 14.5 kDa protein with 19 aa signal peptide. Gen Bank comparison of ricinusin showed weak similarity to a new type of histidine rich antimicrobial protein with unique HEAHEAHEA repeat from Boophilus microplus and Amblyomma hebraeum. The differential gene expression was strongly induced by blood feeding in larva, nymph and adults. The site of differential expression in salivary glands and gut indicates that ricinusin is induced as a part of broad-spectrum immune response. To study the amtimicrobial capacity of ricinusin and its capatibility in clearing Borrelia burgdorferi infections, it was expressed as a His-tagged fusion protein in bacterial expression system. Analysis of the genomic organization of ricinusin gene showed that it is intronless.
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