title: Asparaginyl endopeptidase from the carcinogenic liver fluke, Opisthorchis viverrini, and its potential for serodiagnosis.
abstract: OBJECTIVE
  To isolate and characterize an asparaginyl endopeptidase from the carcinogenic liver fluke, Opisthorchis viverrini, and evaluate its expression profile, biochemical activity, and potential as an immunodiagnostic antigen.
  
  
  METHODS
  The full length mRNA encoding an asparaginyl endopeptidase (family C13), Ov-aep-1, was isolated by immunoscreening of a cDNA bacteriophage library of adult O. viverrini using sera from patients infected with O. viverrini. Investigation of Ov-aep-1 transcripts in developmental stages of the parasite, and phylogenetic analysis, immunohistochemical localization, and recombinant protein expression and enzymology were employed to characterize the Ov-AEP-1 protein. Immunoblotting was used to assess the potential of this enzyme for immunodiagnosis of human opisthorchiasis.
  
  
  RESULTS
  Ov-AEP-1 is characteristic of the C13 cysteine protease family. Ov-aep-1 transcripts were detected in adult and juvenile worms, eggs, and metacercariae. Phylogenetic analysis indicated that Ov-AEP-1 is closely related to homologous proteins in other trematodes. Recombinant Ov-AEP-1 was expressed in bacteria in inclusion bodies and refolded to a soluble form. Excretory-secretory (ES) products derived from adult O. viverrini and refolded recombinant Ov-AEP-1 both displayed catalytic activity against the diagnostic tripeptide substrate, Ala-Ala-Asn-aminomethylcoumarin. Rabbit antiserum raised to recombinant Ov-AEP-1 identified the native AEP-1 protease in both somatic extract and ES products of adult worms. Anti-Ov-AEP-1 IgG immunolocalized the anatomical site of expression to the gut of the fluke, implying a physiological role in digestion of food or activation of other digestive enzymes. Recombinant Ov-AEP-1 was recognized by serum antibodies from patients with opisthorchiasis but not other helminth infections, with a sensitivity and specificity of 85% and 100%, respectively. The positive and negative predictive values are 100% and 67%, respectively.
  
  
  CONCLUSIONS
  The liver fluke, O. viverrini, has a gut-localized asparaginyl endopeptidase. Refolded recombinant Ov-AEP-1 is catalytically active and has potential for immunodiagnosis of human opisthorchiasis.
authors.affiliation: Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand. thewa_la@kku.ac.th
authors.forename: Thewarach
authors.initials: T
authors.lastname: Laha
authors.forename: Jittiyawadee
authors.initials: J
authors.lastname: Sripa
authors.forename: Banchob
authors.initials: B
authors.lastname: Sripa
authors.forename: Mark
authors.initials: M
authors.lastname: Pearson
authors.forename: Leon
authors.initials: L
authors.lastname: Tribolet
authors.forename: Sasithorn
authors.initials: S
authors.lastname: Kaewkes
authors.forename: Paiboon
authors.initials: P
authors.lastname: Sithithaworn
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Brindley
authors.forename: Alex
authors.initials: A
authors.lastname: Loukas
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000909:Antibodies, Helminth / Q000097:blood*; D000947:Antigens, Helminth / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D015723:Gene Library; D015801:Helminth Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009889:Opisthorchiasis / Q000175:diagnosis* / Q000276:immunology / Q000469:parasitology; D009891:Opisthorchis / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development / Q000276:immunology; D010270:Parasite Egg Count; D010802:Phylogeny; D011237:Predictive Value of Tests; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D012698:Serologic Tests; D000099240:Asparaginyl Endopeptidase
chemical_list: D000909:Antibodies, Helminth; D000947:Antigens, Helminth; D003546:Cysteine Endopeptidases; D015801:Helminth Proteins; D011994:Recombinant Proteins; D000099240:Asparaginyl Endopeptidase