A NOVEL KININASE IS PRESENT IN Fasciola hepatica EXCRETION/SECRETION PRODUCTS
Berasain, P.1, Bohrer, C.2, Mandaji, M.3, Reck, J. Jr2, Carmona, C.1, Goñi, F.4, Tarso Benigno Ledur Kist3 and Termignoni, C.2.
1Unidad de Biología Parasitaria, Dpto. de Biología Celular y Molecular, Facultad de Ciencias, Universidad de La Republica Oriental del Uruguay. 2Centro de Biotecnologia e Departamento de Bioquímica, Universidade Federal do Rio Grande do Sul. 3Departamento de Biofísica, Universidade Federal do Rio Grande do Sul. 4Cátedra de Inmunología, Facultad de Química, UDELAR and Dept of Neurology New York University.
The cysteineproteinases play a number of roles in the biology of the parasite trematode F. hepatica such as nutrition, tissue migration and evasion of the host immune responses. Here we describe for the first time a novel kininase activity in the excretion/secretion products (ESP) of the adult liver flukes. By using the guinea pig ileum bioassay we demonstrated ESP are able to efficiently hydrolyze bradykinin (BK, Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg), inactivating the biopeptide. The kininase activity found was completely inhibited by E-64 and not by the metalloproteinase inhibitor EDTA, indicating that the proteolytic enzyme/s responsible for bradykinin inactivation is(are) cysteineproteinase(s). Analysis of the BK cleavage products by capillary electrophoresis showed BK1-6 is the only fragment released after ESP activity, revealing the Ser6-Pro7 bond as the cleavage site. Indeed, this is a unique specificity, since none of the known kininases cleave BK at this site. Surprisingly, ESP do not hydrolyze a fluorogenic analog of BK, o-aminobenzoyl-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Gln-(ethylendiamine 2,4-dinitrophenyl). Because cathepsin L1 is the main liver fluke cysteineproteinase in the adult ESP, we tested recombinant cathepsin, and no kininase activity was detected. Thus, besides CL1, ESP has another cysteineproteinase that is responsible for this novel kininase activity. Probably, this kininase might allow the parasite to interfere with the host inflammatory response modulated by BK through B2 receptors. Acknowledgments: CNPq, FINEP, PRONEX, CAPES and AMSUD-Pasteur.
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