Die chronische Pankreatitis (CP) ist eine wiederkehrende, entzündliche Erkrankung des Pankreas. In den letzten Jahren wurden mehrere Kandidatengene, die zur Entstehung einer CP prädisponieren, identifiziert. Zu diesen Genen gehören PRSS1, PRSS2, SPINK1, CFTR und CTRC. Der Pathogenese der genetisch bedingten CP scheint dabei eine frühzeitige, intrapankreatische Aktivierung von Trypsin zugrunde zu liegen. Cathepsin B (CTSB), eine in Lysosomen vorkommenden Protease, ist in der Lage Trypsinogen zu aktivieren. Genetisch zeigte sich eine Assoziation der p.L26V Variante bei tropisch-kalzifizierender CP, welche bei idiopathischer CP nicht bestätigt wurde. Neben CTSB ist CTSL die am zweithäufigsten vorkommende lysosomale Protease. Funktionelle Untersuchungen zeigten, dass CTSL ein inaktives Trypsin freisetzt. Im Mausmodell zeigten sich bei Ctsl-/- Tieren bei experimentell induzierter Pankreatitis zwei Effekte. Zum einen war die Trypsinaktivität erhöht, zum anderen verlief die Pankreatitis milder, da vermehrt Apoptose anstelle von Nekrose der Azinuszellen auftrat. In dieser Studie wurde mittels uni-direktionaler DNA-Sequenzierung das gesamte CTSL1 untersucht. Dabei fanden wir insgesamt drei seltene nicht-synonyme Varianten. Die Variante c.5A>C (p.N2T, rs112682750) fanden wir bei einem Patienten, wobei diese Variante bereits bei Kontrollen beschrieben wurde. Die Varianten c.126+1G>A und c.915A>C (p.E305D) lagen bei jeweils einer Kontrolle vor. Sowohl seltene als auch häufige Varianten und die berechneten Haplotypen zeigten keinen signifikanten Verteilungsunterschied zwischen Patienten und Kontrollen. Demnach besteht keine Assoziation von Varianten des CTSL1 und CP.

Die chronische Pankreatitis (CP) ist eine wiederkehrende, entzündliche Erkrankung des Pankreas. In den letzten Jahren wurden mehrere Kandidatengene, die zur Entstehung einer CP prädisponieren, identifiziert. Zu diesen Genen gehören PRSS1, PRSS2, SPINK1, CFTR und CTRC. Der Pathogenese der genetisch bedingten CP scheint dabei eine frühzeitige, intrapankreatische Aktivierung von Trypsin zugrunde zu liegen. Cathepsin B (CTSB), eine in Lysosomen vorkommenden Protease, ist in der Lage Trypsinogen zu aktivieren. Genetisch zeigte sich eine Assoziation der p.L26V Variante bei tropisch-kalzifizierender CP, welche bei idiopathischer CP nicht bestätigt wurde. Neben CTSB ist CTSL die am zweithäufigsten vorkommende lysosomale Protease. Funktionelle Untersuchungen zeigten, dass CTSL ein inaktives Trypsin freisetzt. Im Mausmodell zeigten sich bei Ctsl-/- Tieren bei experimentell induzierter Pankreatitis zwei Effekte. Zum einen war die Trypsinaktivität erhöht, zum anderen verlief die Pankreatitis milder, da vermehrt Apoptose anstelle von Nekrose der Azinuszellen auftrat. In dieser Studie wurde mittels uni-direktionaler DNA-Sequenzierung das gesamte CTSL1 untersucht. Dabei fanden wir insgesamt drei seltene nicht-synonyme Varianten. Die Variante c.5A>C (p.N2T, rs112682750) fanden wir bei einem Patienten, wobei diese Variante bereits bei Kontrollen beschrieben wurde. Die Varianten c.126+1G>A und c.915A>C (p.E305D) lagen bei jeweils einer Kontrolle vor. Sowohl seltene als auch häufige Varianten und die berechneten Haplotypen zeigten keinen signifikanten Verteilungsunterschied zwischen Patienten und Kontrollen. Demnach besteht keine Assoziation von Varianten des CTSL1 und CP.

The proteinase, cathepsin L, has been strongly implicated in the processes of tumour invasion and metastasis. A new purification method, three-phase partitioning, characterised in terms of the parameters which affected its fractionation of proteins, was found to simplify the purification of cathepsin L from sheep liver. This method, together with a novel cation-exchange step on S-Sepharose and molecular exclusion chromatography, enabled the enzyme to be purified to homogeneity, in a single-chain form. A further enzyme fraction was isolated as a proteolytically active complex with the endogenous inhibitor of cysteine proteinases, cystatin. Studies on the proteolytically active complex revealed that approximately 60% of it was covalently bound and proteolytically active, while the other 40% was non-covalently bound and proteolytically inactive, in the manner normally found for the binding of cystatin to cysteine proteinases. A cystatin fraction from sheep liver containing variants of cystatin B, was shown to be able to form complexes with free cathepsin L in vitro in a pH-dependent, rapid process, which was mildly stimulated by a reducing agent. Cathepsin L was also isolated from human spleen, but only as a protcolytically inactive complex, presumably also with cystatin(s). The complexed and free cathepsin L from sheep liver were analysed for their pH-dependent characteristics, and it was found that both forms of the enzyme were more active and stable at, or near, neutral pH, than would have been expected from published values. Specific polyclonal antibodies to pure sheep cathepsin L were raised in rabbits and chickens. The chicken egg yolk antibodies were of a much higher titre and were immunoinhibitory towards the enzyme, which the rabbit antibodies were not. Anti-peptide antibodies, raised in rabbits against a peptide sequence selected from the active site of human cathepsin L, were highly specific for cathepsin L and immunoinhibitory towards the enzyme. Together with the polyclonal anti-cathepsin L antibodies, they show promise for immunoinhibitory and immunocytochemical studies on the enzyme, and as potential anti-tumour drugs. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1990.

Cathepsin L, which has been implicated in many tissue degradative pathologies by virtue of its ability to degrade extracellular matrix components, was isolated by a novel, scaled-up protein purification method and purified to homogeneity in the single-chain form. In addition, the high molecular weight variant of cathepsin L covalently complexed with stefin B was isolated. Both cathepsin L and the complex were stable, in respect of their proteolytic activity, to the chaotropic agent urea, both showing enhanced activity in the presence of urea. Urea did not dissociate the complex. The suitability of cathepsin L for a purported extracellular role was addressed by investigating its pH optimum and pH stability. Cathepsins L and B are affected by ionic strength and so buffers of constant ionic strength (rather than constant molarity, and therefore varying ionic strength) were used in determining their pH optima and stability. Cathepsins L and B had apparent pH optima of pH 6.5 and 7.5, respectively, (measured with synthetic substrates) and, contrary to the previous belief, were substantially stable at physiological pH. In Hanks' balanced salt solution, a model of the extracellular fluid, they were shown to be active and stable, cathepsin L having a half-life of 179 s at pH 7.2 and 657 s at pH 6.8 (the peritumour pH). It was also shown that prior reductive activation of these enzymes increased their stability to extracellular conditions, supporting the hypothesis that the active site thiolate-imidazolium ion pair contributes to their stability. The nature of the bond between cathepsin L and stefin B in the covalent complex was examined, using CNBr cleavage, HPLC and amino acid sequencing. Stefin B was shown to be associated with residues 1-137 of cathepsin L via a reduction sensitive linkage which was deduced to be a thioester bond betwen Asp-71 of cathepsin L and Cys-3 of stefin B. Polyclonal antibodies to cathepsin L and stefin B-complexed cathepsin L were raised in rabbits and chickens, and characterised with respect to their suitability for immunocytochemical localisation of these forms of cathepsin L. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1998.

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Our experimental organism Fascioloides magna is a digenetic liver fluke from Fasciolidae family which parasitizes in domestic and free-living ruminants of North America and Central Europe including Czech Republic. In Czech Republic this highly pathogenic worm causes a severe liver damage to cervids and bovids and the prevalence locally reaches up to 95%. The biology of F. magna including e.g. the characteristics of host-parasite molecular interaction and the functions of particular molecules produced by the parasite are not fully understood. According to results of our previous research the excretory-secretory products of F. magna adults contain number of molecules which play the crucial role in host tissue invasion, digestion and evasion of the host immune response. One of the most abundant is cysteine peptidase cathepsin L (FmCL). FmCL is supposed to play various key roles in biological processes of all stages during a life cycle and therefore we can suppose its different expression level in particular life stages. In order to define the expression level of FmCL we performed the pilot study with miracidia and adults where the qPCR method was applied. The results of this experiment revealed much higher expression level of FmCL1 in adults than in miracidia. The attempt to in situ localize the mRNA...

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...

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...