Publication details
Molecular and biochemical characterization of cysteine peptidase inhibitor from Eudiplozoon nipponicum (Monogenea)
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| Year of publication | 2016 |
| Type | Conference abstract |
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| Citation | |
| Description | Inhibitors of cysteine peptidases (cystatins) are proteins produced by a wide range of organisms, including parasites. Besides regulation of the basic physiological functions of parasites they may act either as modulators of the host immune system or regulators of blood-digestion. We identified that cystatin genes are expressed and their protein form probably secreted by Eudiplozoon nipponicum. In following experiments we focused on molecular and functional characterization of cystatin from E. nipponicum (Monogenea). We performed bacterial expression of E. nipponicum cystatin gene (pET19b plasmid vector, E. coli BL21DE3 RIPL cells) and purified the recombinant protein [1]. Phylogenetic trees inferred using Bayesian inference, maximum likelihood, neighbour-joining methods revealed close phylogenetic relationship with cystatins of cestodes. The inhibitory properties and stability of cystatin was fluorometrically measured by adoption of fluorogenic peptide substrate (FR-AMC) and recombinant cysteine peptidases (mouse cathepsin L and cathepsin L3 from E. nipponicum). Successful inhibition of cathepsin L3 from E. nipponicum, probably major molecule responsible for host blood degradation by this monogenean parasite [2], lead us to presumption of cystatin possible role in regulation of this process. Succeeding assays showed that cystatin inhibits the digestion of haemoglobin caused by soluble crude extract from E. nipponicum, its E/S products and recombinant cathepsin L3. Presence of cystatin was successfully detected in both soluble crude extract and excretory-secretory products from E. nipponicum by using anti-cystatin antisera generated in mice and rabbits. Sera were used also as primary antibodies in the immunohistochemical detection of cystatin on cryosections and paraformaldyhyde-fixed paraffin sections of E. nipponicum. In order to test the immunomodulatory potential of cystatin we also carried out a preliminary study by using in vitro cultures of porcine alveolar macrophages (PAMs) stimulated by pathogenic bacteria Haemophilus parasuis which triggers the expression of proinflammatory cytokines by infected PAMs. qPCR analysis showed moderate downregulation of cytokines IL-1 and TNF caused by cystatin of E. nipponicum. |