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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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Kunitz-type inhibitors in Eudiplozoon nipponicum
Černíková, Markéta ; Mikeš, Libor (advisor) ; Jedličková, Lucie (referee)
Proteins containing Kunitz domain are mostly inhibitors of serine proteases. Their general characteristic is the presence of three disulfide bonds and small sizes around 6-10 kDa, although sometimes they consist of several Kunitz domains or they are part of more complex proteins. Their function is usually related to the regulation of physiological and proteolytic processes, but also to an interaction with pathogens or other defense mechanisms, such as being part of the sea anemone mucus or the venom of snakes and other invertebrates. We focused on Kunitz proteins in Eudiplozoon nipponicum, a helminth of the class Monogenea parasiting on gills of common carp (Cyprinus carpio). In the transcriptome of this parasite, several sequences with Kunitz domain have been identified based on similarities with the one already described Kunitz protein, EnKT1, suggesting that this parasite, like other bloodfeeding parasites, uses a whole set of these serine protease inhibitors with other specific functions. Several sequences with the Kunitz domain found in the transcriptome were verified by PCR and optionally supplemented by RACE-PCR. One protein, called EnKC1, was subsequently produced by recombinant expression in E. coli cells of SHuffleTM and Rosetta Gami B strains. Recombinant protein with the Kunitz domain...
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Bioactive molecules involved in blood processing by haematophagous monogeneans of the family Diplozoidae
Jedličková, Lucie
Monogeneans from the family Diplozoidae (subclass Heteronchoinea) are bloodfeeding ectoparasites inhabiting gills of common carp. Digestion of blood in diplozoids is an intracellular process taking place in gut cells within lysosomal cycle in the presence of parasite's peptidases. However, information about the blood digestion comes only from ultrastructural and histochemical analyses. Therefore, I have focused in this work on biochemical and molecular characteristics of bioactive molecules which may participate in blood processing by E. nipponicum adults, especially cysteine peptidases of cathepsin L- and B- types, aspartic peptidases of cathepsin D-type, and Kunitz-type inhibitors of serine peptidases. In homogenates and excretory/secretory (E/S) products of E. nipponicum adults, an activity of cysteine peptidases of cathepsins L-type dominated, followed by an activity of cathepsin D-like aspartic peptidases and a minor cathepsin B-like activity. Inhibitors of the abovementioned peptidase types completely blocked hemoglobinolytic activity in the samples. In the transcriptome of E. nipponicum adults, ten cathepsin L-coding transcripts were found and only one cathepsin B-coding transcript. Primary structures of the encoded enzymes were bioinformatically and phylogenetically compared. Two abundant...
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Bioactive molecules involved in blood processing by haematophagous monogeneans of the family Diplozoidae
Jedličková, Lucie
Monogeneans from the family Diplozoidae (subclass Heteronchoinea) are bloodfeeding ectoparasites inhabiting gills of common carp. Digestion of blood in diplozoids is an intracellular process taking place in gut cells within lysosomal cycle in the presence of parasite's peptidases. However, information about the blood digestion comes only from ultrastructural and histochemical analyses. Therefore, I have focused in this work on biochemical and molecular characteristics of bioactive molecules which may participate in blood processing by E. nipponicum adults, especially cysteine peptidases of cathepsin L- and B- types, aspartic peptidases of cathepsin D-type, and Kunitz-type inhibitors of serine peptidases. In homogenates and excretory/secretory (E/S) products of E. nipponicum adults, an activity of cysteine peptidases of cathepsins L-type dominated, followed by an activity of cathepsin D-like aspartic peptidases and a minor cathepsin B-like activity. Inhibitors of the abovementioned peptidase types completely blocked hemoglobinolytic activity in the samples. In the transcriptome of E. nipponicum adults, ten cathepsin L-coding transcripts were found and only one cathepsin B-coding transcript. Primary structures of the encoded enzymes were bioinformatically and phylogenetically compared. Two abundant...
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Bioactive molecules involved in blood processing by haematophagous monogeneans of the family Diplozoidae
Jedličková, Lucie ; Mikeš, Libor (advisor) ; Horn, Martin (referee) ; Sojka, Daniel (referee)
Monogeneans from the family Diplozoidae (subclass Heteronchoinea) are bloodfeeding ectoparasites inhabiting gills of common carp. Digestion of blood in diplozoids is an intracellular process taking place in gut cells within lysosomal cycle in the presence of parasite's peptidases. However, information about the blood digestion comes only from ultrastructural and histochemical analyses. Therefore, I have focused in this work on biochemical and molecular characteristics of bioactive molecules which may participate in blood processing by E. nipponicum adults, especially cysteine peptidases of cathepsin L- and B- types, aspartic peptidases of cathepsin D-type, and Kunitz-type inhibitors of serine peptidases. In homogenates and excretory/secretory (E/S) products of E. nipponicum adults, an activity of cysteine peptidases of cathepsins L-type dominated, followed by an activity of cathepsin D-like aspartic peptidases and a minor cathepsin B-like activity. Inhibitors of the abovementioned peptidase types completely blocked hemoglobinolytic activity in the samples. In the transcriptome of E. nipponicum adults, ten cathepsin L-coding transcripts were found and only one cathepsin B-coding transcript. Primary structures of the encoded enzymes were bioinformatically and phylogenetically compared. Two abundant...
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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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