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Functional characterization of selected Kunitz proteins of Eudiplozoon nipponicum
Tymich, Alexandr ; Mikeš, Libor (advisor) ; Kašný, Martin (referee)
Proteins containing the Kunitz domain are mostly 6-10 kDa inhibitors of serine proteases, but in exceptional cases they can also inhibit cysteine and aspartic proteases. The main characteristic is the presence of six cysteine residues forming three disulfide bridges creating a typical active loop, which is complementary to the active site of various proteases. The specificity of this binding is largely determined by the amino acid in the P1 position. Their functions include the regulation of a number of physiological events based on proteolysis, e.g. the blood coagulation cascade or immune reactions. However, due to their nature, they have also become a powerful tool for parasitic organisms to interact with their host, where they again target proteases involved in the host's physiological events and thus allow the parasite to survive the interaction with the host. Until recently, representatives of the class Monogenea were a neglected group from the point of view of molecular parasite-host interactions, and only a few works were devoted to their biochemistry and the description of biologically active molecules. In this work, I focused on two selected Kunitz proteins in Eudiplozoon nipponicum, a blood-sucking ectoparasite from the Monogenea class, which has become a fairly common parasite of common...
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Anticoagulation factors and blood uptake by monogeneans of the family Diplozoidae
Skipalová, Karolína ; Mikeš, Libor (advisor) ; Sojka, Dan (referee)
For the successful food intake by organisms that feed on blood is essentials presence of antihaemostatic molecules such as vasodilators, anticoagulant molecules and apyrases., Although members of family Diplozoidae (Heteronchoinea) are blood-feeding parasites on the gills of the fish, these molecules, that could disrupt host hemostasis, have not yet been identified. Thus, the aim of this study was to find molecules with potential anticoagulant activity in homogenates of whole worm bodies and excretory/secretory products of the members of family Diplozoidae. Furthermore perform bioinformatics analysis of sequences obtained from transcriptom project of Eudiplozoon nipponicum (Heteronchoinea: Diplozoidae) and selected proteins (protein domain) then expressed in a recombinant form. We tested inhibitory activity in excretory-secretory products and homogenates of members family Diplozoidae towards coagulation factors IIa and Xa and their specific fluorogenic with 4 negative and 1 positive results. From the results of two transcriptome analysis we discovered three protein families of potential anticoagulants - annexins, serpins and Kunitz-domain proteins. For further analyses we focused on the Kunitz protein family. These proteins contain one or more structurally related active domains which are able to...
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Anticoagulation factors and blood uptake by monogeneans of the family Diplozoidae
Skipalová, Karolína ; Mikeš, Libor (advisor) ; Sojka, Dan (referee)
For the successful food intake by organisms that feed on blood is essentials presence of antihaemostatic molecules such as vasodilators, anticoagulant molecules and apyrases., Although members of family Diplozoidae (Heteronchoinea) are blood-feeding parasites on the gills of the fish, these molecules, that could disrupt host hemostasis, have not yet been identified. Thus, the aim of this study was to find molecules with potential anticoagulant activity in homogenates of whole worm bodies and excretory/secretory products of the members of family Diplozoidae. Furthermore perform bioinformatics analysis of sequences obtained from transcriptom project of Eudiplozoon nipponicum (Heteronchoinea: Diplozoidae) and selected proteins (protein domain) then expressed in a recombinant form. We tested inhibitory activity in excretory-secretory products and homogenates of members family Diplozoidae towards coagulation factors IIa and Xa and their specific fluorogenic with 4 negative and 1 positive results. From the results of two transcriptome analysis we discovered three protein families of potential anticoagulants - annexins, serpins and Kunitz-domain proteins. For further analyses we focused on the Kunitz protein family. These proteins contain one or more structurally related active domains which are able to...
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