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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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The development, constitution and functions of the gland apparatus in schistosome cercariae and endopeptidases in its contents
Titlová, Lucie ; Mikeš, Libor (advisor) ; Konečný, Lukáš (referee)
Flukes of the family Schistosomatidae are blood parasites with two-host life cycles involving aquatic snails as intermediate hosts and avian or mammalian definitive hosts. Cercariae, as invasive aquatic stages of schistosomes, enter the lumen of vessels by penetrating the skin of the definitive host. During their short life, cercariae possess a glandular apparatus consisting of three types of glands with external secretion: penetration glands, escape glands, and a head gland. These glands secrete granules containing, among other things, proteolytic enzymes, which play an important role in the process of penetrating the host's skin, but at the same time they occupy many other functions during the life of the cercaria and other life stages of schistosomes. This thesis summarizes basic knowledge about the anatomy, function and development of the gland apparatus of schistosome cercariae and further focuses on the representation of proteolytic enzymes in these glands, specifically endopeptidases. At the end, it briefly compares the different representation of endopeptidases in some members of the family.
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Salivary glycoproteins of bloodsucking arthropods
Sumová, Petra ; Volf, Petr (advisor) ; Mikeš, Libor (referee)
During obtaining their blood meal, bloodsucking arthropods salivate into their host. Bloodsucking arthropods' saliva contains wide array of bioactive macromolecules. Host organism develops antibody response against many of these molecules. Due to interspecies variability in salivary protein composition, detection of antibody response may serve as a marker of the exposure to individual species of bloodsucking arthropods. Host antibody response is mostly elicited by proteins or glycoproteins. Glycoproteins contain one or more oligosaccharide chains attached to the protein. Glycoprotein's antigenicity could be caused by either both parts, or by only the protein, or the sugar part. This fact has to be taken into consideration for choice of the expression system for recombinant glycoprotein synthesis. This work summarizes current knowledge about structure, function and features of salivary glycoproteins in various species of bloodsucking arthropods.
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Peptidases of monogeneans of the family Diplozoidae
Jedličková, Lucie ; Mikeš, Libor (advisor) ; Dvořák, Jan (referee)
The blood processing mechanisms in monogeneans of the subclass Polyopisthocotylea are known from ultrastructural and histochemical analyses only. In contrast to other blood- feeding parasites, just few biochemical and molecular analyses have been done on digestive enzymes in monogeneans. Therefore, we focused on the biochemical and molecular characterization of hydrolytic enzymes (peptidases) in the hematophagous species Paradiplozoon bliccae and Eudiplozoon nipponicum. The presence of the cysteine class peptidases, mainly cathepsin L, in excretory- secretory products and soluble protein extracts of P. bliccae and E. nipponicum we found. Detection was carried out using fluorogenic substrates, specific inhibitors and the labelled probe DCG-04. On the gels / membranes after electrophoresis / blotting we detected bands of approximately size of 35 kDa in the case of both species and 24 kDa for E. nipponicum. Soluble protein extracts of worms were separated by 2D gel electrophoresis and relevant spots around 35 kDa (P. bliccae) and around 25 ˗ 35 kDa (E. nipponicum) were confirmed by mass spectrometry as cathepsins L. Using degenerate primers based on the conserved motifs of cysteine class peptidases, a partial sequence of cathepsin L gene from E. nipponicum was obtained. Furthermore, 3'RACE PCR method...
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Glycocalyx shedding by cercariae of bird schistosomes
Chaloupecká, Jana ; Mikeš, Libor (advisor) ; Štěrba, Ján (referee)
Trichobilharzia spp. are avian schistosomes related to medically important human parasites of the genus Schistosoma. Penetrating cercariae are well known as causative agent of cercarial dermatitis in humans. Cercariae actively penetrate the skin of definitive hosts and transform into schistosomula. This process is preceded by cercarial tail detachment and includes emptying of penetration glands and extensive surface changes. One of these changes is the loss of highly immunogenic glycocalyx which represents a protective coat in the aquatic environment. The glycocalyx has specific composition of saccharide molecules which are bound to lipids or proteins on the membrane of cercarial tegument. There is only limited information about the mechanism of shedding. Hypotheses based on indirect evidences suggest that peptidases or (phospho)lipases from penetration glands could be involved. This work describes the changes in surface glycosylation during transformation of cercariae into schistosomula by fluorescently labelled lectins and monoclonal antibodies against Lewis X antigen. Lectins UEA-I, LTA and PNA have been chosen as markers of transformation of T. regenti. Further, our experiments have been focused on shedding of cercarial glycocalyx. During in vitro induction of penetration gland emptying and...
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Features and functions of glycocalyx of trematode cercariae
Chaloupecká, Jana ; Mikeš, Libor (advisor) ; Kašný, Martin (referee)
Trematodes are parasites from phylum Platyhelminthes which have compex life cycles involving two to four hosts. This work focuses especially on trematodes of the family Schistosomatidae. Their cercariae which leave the snail intermediate host, actively penetrate the skin of definitive hosts and transform into schistosomula. This is accompanied by detachment of cercarial tail and emptying of penetration glands. During transformation, cercarial bodies undergo extensive ultrastructural and molecular changes. One of these changes is the loss of surface glycocalyx which represents a protective coat in the aquatic environment. In glycocalyx shedding, participation of proteolytic enzymes from cercarial penetration glands is expected during invasion of the host. Glycocalyx has specific composition of saccharide molecules which are bound to lipids or proteins on the membrane of cercarial tegument. This work describes the origin, ultrastructure, saccharide composition, function and shedding mechanism of cercarial glycocalyx.
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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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Cathepsins L of Diplostomum pseudospathaceum cercariae
Perháčová, Terézia ; Mikeš, Libor (advisor) ; Hartmann, David (referee)
This study is focused on cercarial cysteine peptidases of the trematode Diplostomum pseudospathaceum. It follows previous research which confirmed the presence of a 24kDa cysteine peptidase in cercariae biochemically and by mass spectrometry. It was postulated, that the function of this peptidase is histolytic, when cercariae penetrate the tissues. During an attempt to purify this peptidase and characterize its peptidolytic activity, it was found out that the cercarial homogenate containsmore different peptidases varying in their pI. Tests of peptidolytic activity and inhibition have shown that these peptidases are cathepsin L-like. They are active over a broad spectrum of pH with optima of activities in weakly acidicor neutral pH. Using degenerate primers based on conserved motifs of cysteine pepridases, partial sequences of three genes for cathepsin L of D. pseudospataceum (DpCL1, 2 a 3) were obtained. Then the complete sequences of DpCL2 and 3 genes and partial sequence (without 5'end) of DpCL1 were obtained by RACE PCR. To confirm function of these peptidases we tried to immunolocalize them. We assumed that they are localized in penetration glands. Preliminary results suggested that some of the cathepsins could be also localized in the gut of cercariae. For more detailed biochemical...
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