title: Molecular and immunological characterization of cathepsin L-like cysteine protease of Paragonimus pseudoheterotremus.
abstract: Cathepsin L is a cysteine protease belonging to the papain family. In parasitic trematodes, cathepsin L plays essential roles in parasite survival and host-parasite interactions. In this study, cathepsin L of the lung fluke Paragonimus pseudoheterotremus (PpsCatL) was identified and its molecular biological and immunological features characterized. A sequence analysis of PpsCatL showed that the gene encodes a 325-amino-acid protein that is most similar to P. westermani cathepsin L. The in silico three-dimensional structure suggests that PpsCatL is a pro-enzyme that becomes active when the propeptide is cleaved. A recombinant pro-PpsCatL lacking the signal peptide (rPpsCatL), with a molecular weight of 35 kDa, was expressed in E. coli and reacted with P. pseudoheterotremus-infected rat sera. The native protein was detected in crude worm antigens and excretory-secretory products and was localized in the cecum and in the lamellae along the intestinal tract of the adult parasite. Enzymatic activity of rPpsCatL showed that the protein could cleave the fluorogenic substrate Z-Phe-Arg-AMC after autocatalysis but was inhibited with E64. The immunodiagnostic potential of the recombinant protein was evaluated with an enzyme-linked immunosorbent assay (ELISA) and suggested that rPpsCatL can detect paragonimiasis with high sensitivity and specificity (100 and 95.6 %, respectively). This supports the further development of an rPpsCatL-ELISA as an immunodiagnostic tool.
authors: Department of Helminthology, Faculty of Tropical Medicine, Mahidol University, Ratchathewi, Bangkok, 10400, Thailand.
authors: Tippayarat
authors: T
authors: Yoonuan
authors: Department of Helminthology, Faculty of Tropical Medicine, Mahidol University, Ratchathewi, Bangkok, 10400, Thailand.
authors: Supaporn
authors: S
authors: Nuamtanong
authors: Department of Helminthology, Faculty of Tropical Medicine, Mahidol University, Ratchathewi, Bangkok, 10400, Thailand.
authors: Paron
authors: P
authors: Dekumyoy
authors: Department of Helminthology, Faculty of Tropical Medicine, Mahidol University, Ratchathewi, Bangkok, 10400, Thailand.
authors: Orawan
authors: O
authors: Phuphisut
authors: Department of Helminthology, Faculty of Tropical Medicine, Mahidol University, Ratchathewi, Bangkok, 10400, Thailand. poom.adi@mahidol.ac.th.
authors: Poom
authors: http://orcid.org/0000-0002-0313-9900
authors: P
authors: Adisakwattana
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000947:Antigens, Helminth / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D001483:Base Sequence; D056668:Cathepsin L / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D015801:Helminth Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010237:Paragonimiasis / Q000175:diagnosis / Q000469:parasitology*; D010238:Paragonimus / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D051381:Rats; D017208:Rats, Wistar; D016415:Sequence Alignment
chemical_list: D000947:Antigens, Helminth; D015801:Helminth Proteins; D056668:Cathepsin L
keywords: Cathepsin L; Cecum; ELISA; Enzyme-linked immunosorbent assay; Paragonimus pseudoheterotremus; Recombinant protein