Serine peptidases are involved in many physiological processes including digestion, haemostasis and complement cascade. Parasites regulate activities of host serine peptidases to their own benefit, employing various inhibitors, many of which belong to the Kunitz-type protein family. In this study, we confirmed the presence of potential anticoagulants in protein extracts of the haematophagous monogenean Eudiplozoon nipponicum which parasitizes the common carp. We then focused on a Kunitz protein (EnKT1) discovered in the E. nipponicum transcriptome, which structurally resembles textilinin-1, an antihemorrhagic snake venom factor from Pseudonaja textilis. The protein was recombinantly expressed, purified and biochemically characterised. The recombinant EnKT1 did inhibit in vitro activity of Factor Xa of the coagulation cascade, but exhibited a higher activity against plasmin and plasma kallikrein, which participate in fibrinolysis, production of kinins, and complement activation. Anti-coagulation properties of EnKT1 based on the inhibition of Factor Xa were confirmed by thromboelastography, but no effect on fibrinolysis was observed. Moreover, we discovered that EnKT1 significantly impairs the function of fish complement, possibly by inhibiting plasmin or Factor Xa which can act as a C3 and C5 convertase. We localised Enkt1 transcripts and protein within haematin digestive cells of the parasite by RNA in situ hybridisation and immunohistochemistry, respectively. Based on these results, we suggest that the secretory Kunitz protein of E. nipponicum has a dual function. In particular, it impairs both haemostasis and complement activation in vitro, and thus might facilitate digestion of a host's blood and protect a parasite's gastrodermis from damage by the complement. This study presents, to our knowledge, the first characterisation of a Kunitz protein from monogeneans and the first example of a parasite Kunitz inhibitor that impairs the function of the complement.
- MeSH
- antifibrinolytika chemie imunologie MeSH
- antikoagulancia chemie imunologie MeSH
- faktor Xa imunologie MeSH
- hemostáza * MeSH
- infekce červy třídy Trematoda krev imunologie parazitologie veterinární MeSH
- inhibitory enzymů chemie imunologie MeSH
- inhibitory faktoru Xa chemie imunologie MeSH
- interakce hostitele a parazita MeSH
- kapři krev imunologie parazitologie MeSH
- komplement imunologie MeSH
- nemoci ryb krev imunologie parazitologie MeSH
- plasmin imunologie MeSH
- plazmatický kalikrein antagonisté a inhibitory imunologie MeSH
- proteiny červů chemie genetika imunologie MeSH
- sekvence aminokyselin MeSH
- sekvenční seřazení MeSH
- Trematoda chemie genetika imunologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Serpins are a superfamily of serine peptidase inhibitors that participate in the regulation of many physiological and cell peptidase-mediated processes in all organisms (e.g. in blood clotting, complement activation, fibrinolysis, inflammation, and programmed cell death). It was postulated that in the blood-feeding members of the monogenean family Diplozoidae, serpins could play an important role in the prevention of thrombus formation, activation of complement, inflammation in the host, and/or in the endogenous regulation of protein degradation. RESULTS: In silico analysis showed that the DNA and primary protein structures of serpin from Eudiplozoon nipponicum (EnSerp1) are similar to other members of the serpin superfamily. The inhibitory potential of EnSerp1 on four physiologically-relevant serine peptidases (trypsin, factor Xa, kallikrein, and plasmin) was demonstrated and its presence in the worm's excretory-secretory products (ESPs) was confirmed. CONCLUSION: EnSerp1 influences the activity of peptidases that play a role in blood coagulation, fibrinolysis, and complement activation. This inhibitory potential, together with the serpin's presence in ESPs, suggests that it is likely involved in host-parasite interactions and could be one of the molecules involved in the control of feeding and prevention of inflammatory responses.
- MeSH
- DNA helmintů chemie MeSH
- fylogeneze MeSH
- infekce červy třídy Trematoda parazitologie veterinární MeSH
- inhibitory serinových proteinas chemie genetika izolace a purifikace metabolismus MeSH
- kapři parazitologie MeSH
- nemoci ryb parazitologie MeSH
- počítačová simulace MeSH
- polymerázová řetězová reakce MeSH
- rekombinantní proteiny genetika izolace a purifikace metabolismus MeSH
- sekvence aminokyselin MeSH
- sekvence nukleotidů MeSH
- sekvenční seřazení MeSH
- serpiny chemie genetika izolace a purifikace metabolismus MeSH
- Trematoda chemie klasifikace enzymologie genetika MeSH
- žábry parazitologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
