DOI: 10.14457/tu.the.2015.789
title: Molecular and biochemical characterization of type 1 cystatin stefin-2 of the liver fluke Fasciola gigantica

description: Cysteine proteases including cathepsin B and cathepsin L are involved in physiological and biological processes in all living organisms and imbalanced activity of these proteases may cause several diseases.  They are also important antigens in the trematode genus Fasciola and have vital roles in parasite survival including protection, infection and nutrition of the liver fluke.  The biological roles and application of parasite cathepsins, e.g. in chemotherapy and as vaccines have been studied but data concerning their regulation is still incomplete.  Especially, the cysteine protease inhibitors of the cystatin superfamily have not been investigated in depth in trematodes.  This research was conducted to characterize the molecular and biochemical properties of a type 1 cystatin (Stefin-2) in the liver fluke Fasciola gigantica.In the present study, a cDNA encoding type 1 cystatin of the parasite, FgStefin-2, was isolated from total RNA of adult F. gigantica by RT-PCR.  A FgStefin-2-specific probe detected gene and transcript in Southern and Northern analyses of genomic DNA and total RNA of the fluke. FgStefin-2, which unusually for type 1 cystatins carries a signal peptide, was expressed as active recombinant protein in Escherichia coli (rFgStefin-2).  Purified rFgStefin-2 was used for cysteine protease inhibition assays, functional analysis and polyclonal antibody production.  The polyclonal antibody was used to study the distribution of native FgStefin-2 in 2- and 4-week-old juveniles and adult F. gigantica and to detect rFgStefin-2 and native FgStefin-2 in crude worm (CW) extract and excretory/secretory (ES) products of adult F. gigantica in immunoblots.  Immunohistochemical analysis showed that FgStefin-2 is located in several tissues of the parasite including the prostate gland in adults and gut epithelium in all stages.  Immunoblots demonstrated that the polyclonal antibody reacted with rFgStefin-2, CW extract and ES product of the adult parasite, but did not cross-react with rFgMDCd10, rFgStefin-1 and CW extracts of other trematodes.  The inhibitory properties of rFgStefin-2 against bovine cathepsin B, bovine cathepsin L and the proteolytic activity of ES products, CW extracts from metacercariae and adult parasite were characterized by using fluorogenic substrates and by zymography using gelatin as substrate.  The recombinant protein exhibited inhibitory activity against cysteine proteases (cathepsins B and L) and the proteolytic activity of ES products, CW extracts from metacercariae and adult parasites.  This protein was found to be active over a wide pH range and heat stable.  Preliminary study on immunomodulatory properties of the protein showed that it may interfere with an immune response mechanism that is involved in polyclonal T-cell proliferation.  Furthermore, the novel cathepsin B5 of F. gigantica, FgCB5, was expressed and purified in a yeast-based eukaryotic expression system.  Immunoblots showed that procathepsin B5 was detected in CW extract and in minor amounts in ES product.  Analyses of recombinant cathepsin B5 revealed that it was active at acidic pH, able to effectively digest several of the host substrates and also exhibited substantial exopeptidase activity.  Inhibitory activities of rFgStefin-1, rFgStefin-2 and human cystatin C against rFgCB5 demonstrated nanomolar inhibition constants.  This experiment was used to confirm that rFgStefin-2 has been evolutionary adapted to block cathepsin B.  In summary, FgStefin-2 may play important roles, for example in the protection of the minute newly excysted juvenile from autoproteolysis and in the regulation of cysteine protease activity in the reproductive system.  This knowledge may help to evaluate application of the protein as diagnosis tool, drug target and vaccine candidate against fascioliasis.