title: Characterization of a gut-associated asparaginyl endopeptidase of Clonorchis sinensis.
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.
authors.affiliation: Department of Parasitology and Tropical Medicine, and Institute of Health Sciences, Gyeongsang National University School of Medicine, Jinju 660-751, Republic of Korea.
authors.forename: Jung-Mi
authors.initials: JM
authors.lastname: Kang
authors.affiliation: Department of Parasitology and Tropical Medicine, and Institute of Health Sciences, Gyeongsang National University School of Medicine, Jinju 660-751, Republic of Korea.
authors.forename: Jinyoung
authors.initials: J
authors.lastname: Lee
authors.affiliation: Department of Parasitology and Tropical Medicine, and Institute of Health Sciences, Gyeongsang National University School of Medicine, Jinju 660-751, Republic of Korea.
authors.forename: Hye-Lim
authors.initials: HL
authors.lastname: Ju
authors.affiliation: Division of Malaria and Parasitic Diseases, National Institute of Health, Korea Centers for Disease Control and Prevention, Seoul 122-701, Republic of Korea.
authors.forename: Jung Won
authors.initials: JW
authors.lastname: Ju
authors.affiliation: Department of Parasitology, College of Veterinary Medicine, Gyeongsang National University, Jinju 660-701, Republic of Korea.
authors.forename: Jong-Hyun
authors.initials: JH
authors.lastname: Kim
authors.affiliation: Asan Institute for Life Sciences, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736, Republic of Korea.
authors.forename: Jhang Ho
authors.initials: JH
authors.lastname: Pak
authors.affiliation: Department of Tropical Medicine, and Inha Research Institute for Medical Sciences, Inha University School of Medicine, Incheon 400-712, Republic of Korea.
authors.forename: Tong-Soo
authors.initials: TS
authors.lastname: Kim
authors.affiliation: Department of Parasitology and Tropical Medicine, Kyungpook National University School of Medicine, Daegu 700-422, Republic of Korea.
authors.forename: Yeonchul
authors.initials: Y
authors.lastname: Hong
authors.affiliation: Department of Parasitology and Tropical Medicine, and Institute of Health Sciences, Gyeongsang National University School of Medicine, Jinju 660-751, Republic of Korea.
authors.forename: Woon-Mok
authors.initials: WM
authors.lastname: Sohn
authors.affiliation: Department of Parasitology and Tropical Medicine, and Institute of Health Sciences, Gyeongsang National University School of Medicine, Jinju 660-751, Republic of Korea. Electronic address: bkna@gnu.ac.kr.
authors.forename: Byoung-Kuk
authors.initials: BK
authors.lastname: Na
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D003003:Clonorchiasis / Q000469:parasitology*; D003004:Clonorchis sinensis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005260:Female; D041981:Gastrointestinal Tract / Q000201:enzymology; D015801:Helminth Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D051381:Rats; D017207:Rats, Sprague-Dawley; D016415:Sequence Alignment; D000099240:Asparaginyl Endopeptidase
chemical_list: D003546:Cysteine Endopeptidases; D015801:Helminth Proteins; D000099240:Asparaginyl Endopeptidase
keywords: Asparaginyl endopeptidase; Clonorchis sinensis; Intestine; Legumain