Tick saliva injected into the vertebrate host contains bioactive anti-proteolytic proteins from the cystatin family; however, the molecular basis of their unusual biochemical and physiological properties, distinct from those of host homologs, is unknown. Here, we present Ricistatin, a novel secreted cystatin identified in the salivary gland transcriptome of Ixodes ricinus ticks. Recombinant Ricistatin inhibited host-derived cysteine cathepsins and preferentially targeted endopeptidases, while having only limited impact on proteolysis driven by exopeptidases. Determination of the crystal structure of Ricistatin in complex with a cysteine cathepsin together with characterization of structural determinants in the Ricistatin binding site explained its restricted specificity. Furthermore, Ricistatin was potently immunosuppressive and anti-inflammatory, reducing levels of pro-inflammatory cytokines IL-6, IL-1β, and TNF-α and nitric oxide in macrophages; IL-2 and IL-9 levels in Th9 cells; and OVA antigen-induced CD4+ T cell proliferation and neutrophil migration. This work highlights the immunotherapeutic potential of Ricistatin and, for the first time, provides structural insights into the unique narrow selectivity of tick salivary cystatins determining their bioactivity.
- MeSH
- cystatiny * farmakologie MeSH
- cystein metabolismus MeSH
- endopeptidasy metabolismus MeSH
- kathepsiny metabolismus MeSH
- klíště * chemie MeSH
- obratlovci MeSH
- proteasy metabolismus MeSH
- slinné cystatiny chemie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Eudiplozoon nipponicum (Goto, 1891) is a hematophagous monogenean ectoparasite which inhabits the gills of the common carp (Cyprinus carpio). Heavy infestation can lead to anemia and in conjunction with secondary bacterial infections cause poor health and eventual death of the host. This study is based on an innovative approach to protein localization which has never been used in parasitology before. Using laser capture microdissection, we dissected particular areas of the parasite body without contaminating the samples by surrounding tissue and in combination with analysis by mass spectrometry obtained tissue-specific proteomes of tegument, intestine, and parenchyma of our model organism, E. nipponicum. We successfully verified the presence of certain functional proteins (e.g. cathepsin L) in tissues where their presence was expected (intestine) and confirmed that there were no traces of these proteins in other tissues (tegument and parenchyma). Additionally, we identified a total of 2,059 proteins, including 72 peptidases and 33 peptidase inhibitors. As expected, the greatest variety was found in the intestine and the lowest variety in the parenchyma. Our results are significant on two levels. Firstly, we demonstrated that one can localize all proteins in one analysis and without using laboratory animals (antibodies for immunolocalization of single proteins). Secondly, this study offers the first complex proteomic data on not only the E. nipponicum but within the whole class of Monogenea, which was from this point of view until recently neglected.
- MeSH
- kapři parazitologie MeSH
- kathepsiny analýza metabolismus MeSH
- laserová záchytná mikrodisekce MeSH
- parenchymatická tkáň metabolismus MeSH
- ploštěnci metabolismus MeSH
- proteasy analýza metabolismus MeSH
- proteom analýza MeSH
- proteomika metody MeSH
- střevní sliznice metabolismus MeSH
- tandemová hmotnostní spektrometrie MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- žábry parazitologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
To ensure successful feeding tick saliva contains a number of inhibitory proteins that interfere with the host immune response and help to create a permissive environment for pathogen transmission. Among the potential targets of the salivary cystatins are two host cysteine proteases, cathepsin S, which is essential for antigen- and invariant chain-processing, and cathepsin C (dipeptidyl peptidase 1, DPP1), which plays a critical role in processing and activation of the granule serine proteases. Here, the effect of salivary cystatin OmC2 fromOrnithodoros moubatawas studied using differentiated MUTZ-3 cells as a model of immature dendritic cells of the host skin. Following internalization, cystatin OmC2 was initially found to inhibit the activity of several cysteine cathepsins, as indicated by the decreased rates of degradation of fluorogenic peptide substrates. To identify targets, affinity chromatography was used to isolate His-tagged cystatin OmC2 together with the bound proteins from MUTZ-3 cells. Cathepsins S and C were identified in these complexes by mass spectrometry and confirmed by immunoblotting. Furthermore, reduced increase in the surface expression of MHC II and CD86, which are associated with the maturation of dendritic cells, was observed. In contrast, human inhibitor cystatin C, which is normally expressed and secreted by dendritic cells, did not affect the expression of CD86. It is proposed that internalization of salivary cystatin OmC2 by the host dendritic cells targets cathepsins S and C, thereby affecting their maturation.
- MeSH
- antigeny CD86 MeSH
- antigeny diferenciační B-lymfocytární MeSH
- buněčné linie MeSH
- cystatiny metabolismus MeSH
- dendritické buňky imunologie metabolismus MeSH
- epoxidové sloučeniny imunologie metabolismus MeSH
- geny MHC třídy II imunologie MeSH
- kathepsin C metabolismus MeSH
- kathepsiny chemie imunologie metabolismus MeSH
- klíšťata enzymologie MeSH
- lidé MeSH
- lyzozomy enzymologie MeSH
- MHC antigeny II. třídy MeSH
- Ornithodoros enzymologie MeSH
- rekombinantní proteiny MeSH
- sliny enzymologie MeSH
- tyrosin analogy a deriváty imunologie metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Trichobilharzia regenti and T. szidati are schistosomes that infect birds. although T. regenti/T. szidati can only complete their life cycle in specific bird hosts (waterfowl), their larvae-cercariae are able to penetrate, transform and then migrate as schistosomula in nonspecific hosts (e.g., mouse, man). Peptidases are among the key molecules produced by these schistosomes that enable parasite invasion and survival within the host and include cysteine peptidases such as cathepsins B1 and B2. These enzymes are indispensable bio-catalysts in a number of basal biological processes and host-parasite interactions, e.g., tissue invasion/migration, nutrition and immune evasion. Similar biochemical and functional characteristics were observed for cathepsins B1 and B2 in bird schistosomes (T. regenti, T. szidati) and also for their homologs in human schistosomes (Schistosoma mansoni, S. japonicum). Therefore, data obtained in the research of bird schistosomes can also be exploited for the control of human schistosomes such as the search for targets of novel chemotherapeutic drugs and vaccines.
- MeSH
- cerkárie enzymologie MeSH
- kathepsiny chemie genetika metabolismus MeSH
- lidé MeSH
- molekulární sekvence - údaje MeSH
- nemoci ptáků parazitologie MeSH
- ptáci parazitologie MeSH
- Schistosoma enzymologie MeSH
- sekvence aminokyselin MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Cathepsins S and H are present in immune cells and tissues and may play a role in the activation of an adoptive immune response. Our goal was to assess their protein levels in pleural fluids from 82 patients who underwent thoracentesis or thoracoscopy for therapeutic or diagnostic reasons and to relate them to an inflammatory, neoplastic or hemodynamic origin. Pleural effusions were also analyzed for a panel of 13 inflammatory or proliferative markers to test possible links to a nonspecific host reaction. Increased levels of cathepsin S were found in parainflammatory and cancer-related effusions compared to transudates. Cathepsin H levels were elevated only in parainflammatory effusions, whereas the levels in cancer-related effusions were comparable to transudates. Cathepsin S values significantly correlated with LDH, alpha-1-AT, VEGF, sICAM, sVCAM, MPO, uPA, MMP-9/TIMP-1, IL-8 and MCP-1, but not with CRP, IL-10 or cathepsin H. In contrast to cathepsin S, cathepsin H values did not correlate with markers of inflammation, indicating a specific role for cathepsin H in the pleural host response. In conclusion, the estimation of cathepsin S and cathepsin H may help to distinguish between effusions of different etiology.
- MeSH
- biologické markery krev metabolismus MeSH
- cysteinové endopeptidasy krev metabolismus MeSH
- kathepsin H MeSH
- kathepsiny krev metabolismus MeSH
- lidé středního věku MeSH
- lidé MeSH
- maligní pleurální výpotek enzymologie MeSH
- nádorové biomarkery krev metabolismus MeSH
- nádory plic enzymologie MeSH
- paraneoplastické syndromy enzymologie MeSH
- pleurální výpotek enzymologie MeSH
- pleuritida enzymologie MeSH
- senioři MeSH
- zánět enzymologie MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- práce podpořená grantem MeSH
- MeSH
- interakce hostitele a parazita fyziologie genetika MeSH
- kathepsin B MeSH
- kathepsiny genetika izolace a purifikace metabolismus MeSH
- larva parazitologie MeSH
- paraziti MeSH
- Pichia genetika metabolismus MeSH
- ptáci parazitologie MeSH
- Schistosoma japonicum genetika metabolismus parazitologie MeSH
- Schistosoma mansoni genetika metabolismus parazitologie MeSH
- Schistosomatidae metabolismus MeSH
Free propeptides are known to function as inhibitors of the parental mature cysteine cathepsins. This general rule, however, does not apply to the aminopeptidase cathepsin H. Screening of propeptide fragments for their inhibitory potency revealed no significant effect on the native mature cathepsin H. On the other hand, inhibitory interaction was established with recombinant cathepsin H that displays endopeptidase activity due to a lack of the mini-chain. This finding suggests that the propeptide-binding region is structurally rearranged during maturation processing and mini-chain formation, which impairs the effective recognition of mature cathepsin H by its own propeptide.
- MeSH
- aktivace enzymů MeSH
- cirkulární dichroismus MeSH
- cysteinové endopeptidasy chemie metabolismus MeSH
- financování organizované MeSH
- kathepsiny antagonisté a inhibitory chemie metabolismus MeSH
- molekulární modely MeSH
- molekulární sekvence - údaje MeSH
- peptidy chemie metabolismus MeSH
- prekurzory enzymů chemie metabolismus MeSH
- sekvence aminokyselin MeSH
- terciární struktura proteinů MeSH
- MeSH
- kathepsiny metabolismus MeSH
- remodelace kosti MeSH
- resorpce kosti MeSH
- Publikační typ
- kongresy MeSH
