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Experimental parasitology

Characterization of a gut-associated asparaginyl endopeptidase of Clonorchis sinensis.


PMID 25819296

Abstract

Asparaginyl endopeptidases (AEP: EC 3.4.22.34) are a family of cysteine proteases classified into the MEROPS clan CD, family C13. In this study, we characterized the biochemical and antigenic properties of an AEP of Clonorchis sinensis (CsAEP). The recombinant CsAEP showed hydrolytic activity at pH values ranging from acidic to neutral with optimum activity at pH 6.0. While the recombinant CsAEP was stable at neutral pHs, it was unstable at acidic pHs and resulted in loss of enzymatic activity. The recombinant enzyme was effectively inhibited by iodoacetic acid and N-ethylmaleimide, but not by E-64. The partially purified native CsAEP showed biochemical properties similar to the recombinant enzyme. Native CsAEP is likely to be cleaved into an N-terminal mature enzyme and a C-terminal fragment via autocatalytic activation at acidic pHs. Polyclonal antibody raised against the recombinant CsAEP recognized three forms of CsAEP, proenzyme, the N-terminal mature enzyme and the C-terminal fragment, in the worm extract (WE) of C. sinensis. However, only the C-terminal fragment was mainly found in the excretory and secretory (ES) products of the parasite. Strong CsAEP activity was found in the WE, but only a trace level of CsAEP activity was detected in the ES products of the parasite. CsAEP was expressed in various developmental stages of C. sinensis, from metacercariae to adults, and was found to be localized in the intestine of the parasite as well as in intestinal contents. Sera from rats experimentally infected with C. sinensis reacted with CsAEP beginning 4 weeks after infection. These results suggest that CsAEP is a gut-associated enzyme synthesized in the intestine of C. sinensis and subsequently secreted into the intestinal lumen of the parasite.

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